Pyrrolobenzodiazepines and targeted conjugates

ABSTRACT

A compound, or a pharmaceutically acceptable salt or solvate thereof, or conjugates thereof, selected from the group consisting of: 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     wherein:
         (a) R 10  is H, and R 11  is OH, OR A , where R A  is saturated C 1-4  alkyl;   (b) R 10  and R 11  form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; or   (c) R 10  is H and R 11  is SO z M, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation, or both M together are a divalent pharmaceutically acceptable cation.

The present invention relates to pyrrolobenzodiazepines (PBDs), inparticular pyrrolobenzodiazepine dimers having a C2-C3 double bond andan aryl group at the C2 position in each monomer unit, and theirinclusion in targeted conjugates.

BACKGROUND TO THE INVENTION

Some pyrrolobenzodiazepines (PBDs) have the ability to recognise andbond to specific sequences of DNA; the preferred sequence is PuGPu. Thefirst PBD antitumour antibiotic, anthramycin, was discovered in 1965(Leimgruber, et al., J. Am. Chem. Soc., 87, 5793-5795 (1965);Leimgruber, et al., J. Am. Chem. Soc., 87, 5791-5793 (1965)). Sincethen, a number of naturally occurring PBDs have been reported, andnumerous synthetic routes have been developed to a variety of analogues(Thurston, et al., Chem. Rev. 1994, 433-465 (1994); Antonow, D. andThurston, D. E., Chem. Rev. 2011 111 (4), 2815-2864). Family membersinclude abbeymycin (Hochlowski, et al., J. Antibiotics, 40, 145-148(1987)), chicamycin (Konishi, et al., J. Antibiotics, 37, 200-206(1984)), DC-81 (Japanese Patent 58-180 487; Thurston, et al., Chem.Brit., 26, 767-772 (1990); Bose, et al., Tetrahedron, 48, 751-758(1992)), mazethramycin (Kuminoto, et al., J. Antibiotics, 33, 665-667(1980)), neothramycins A and B (Takeuchi, et al., J. Antibiotics, 29,93-96 (1976)), porothramycin (Tsunakawa, et al., J. Antibiotics, 41,1366-1373 (1988)), prothracarcin (Shimizu, et al, J. Antibiotics, 29,2492-2503 (1982); Langley and Thurston, J. Org. Chem., 52, 91-97(1987)), sibanomicin (DC-102)(Hara, et al., J. Antibiotics, 41, 702-704(1988); Itoh, et al., J. Antibiotics, 41, 1281-1284 (1988)), sibiromycin(Leber, et al., J. Am. Chem. Soc., 110, 2992-2993 (1988)) and tomamycin(Arima, et al., J. Antibiotics, 25, 437-444 (1972)). PBDs are of thegeneral structure:

They differ in the number, type and position of substituents, in boththeir aromatic A rings and pyrrolo C rings, and in the degree ofsaturation of the C ring. In the B-ring there is either an imine (N═C),a carbinolamine (NH—CH(OH)), or a carbinolamine methyl ether(NH—CH(OMe)) at the N10-C11 position which is the electrophilic centreresponsible for alkylating DNA. All of the known natural products havean (S)-configuration at the chiral C11a position which provides themwith a right-handed twist when viewed from the C ring towards the Aring. This gives them the appropriate three-dimensional shape forisohelicity with the minor groove of B-form DNA, leading to a snug fitat the binding site (Kohn, In Antibiotics III. Springer-Verlag, NewYork, pp. 3-11 (1975); Hurley and Needham-VanDevanter, Acc. Chem. Res.,19, 230-237 (1986)). Their ability to form an adduct in the minorgroove, enables them to interfere with DNA processing, hence their useas antitumour agents.

It has been previously disclosed that the biological activity of thesemolecules can be potentiated by joining two PBD units together throughtheir C8/C′-hydroxyl functionalities via a flexible alkylene linker(Bose, D. S., et al., J. Am. Chem. Soc., 114, 4939-4941 (1992);Thurston, D. E., et al., J. Org. Chem., 61, 8141-8147 (1996)). The PBDdimers are thought to form sequence-selective DNA lesions such as thepalindromic 5′-Pu-GATC-Py-3′ interstrand cross-link (Smellie, M., etal., Biochemistry, 42, 8232-8239 (2003); Martin, C., et al.,Biochemistry, 44, 4135-4147) which is thought to be mainly responsiblefor their biological activity. One example of a PBD dimmer, SG2000(SJG-136):

has recently entered Phase II clinical trials in the oncology area(Gregson, S., et al., J. Med. Chem., 44, 737-748 (2001); Alley, M. C.,et al., Cancer Research, 64, 6700-6706 (2004); Hartley, J. A., et al.,Cancer Research, 64, 6693-6699 (2004)).

More recently, the present inventors have previously disclosed in WO2005/085251, dimeric PBD compounds bearing C2 aryl substituents, such asSG2202 (ZC-207):

and in WO2006/111759, bisulphites of such PBD compounds, for exampleSG2285 (ZC-423):

These compounds have been shown to be highly useful cytotoxic agents(Howard, P. W., et al., Bioorg. Med. Chem. (2009), 19 (22), 6463-6466,doi: 10.1016/j.bmc1.2009.09.012).

Due to the manner in which these highly potent compounds act incross-linking DNA, these molecules have been made symmetrically. Thisprovides for straightforward synthesis, either by constructing the PBDmoieties simultaneously having already formed the dimer linkage, or byreacting already constructed PBD moieties with the dimer linking group.

WO 2010/043880 discloses unsymmetrical dimeric PBD compound bearing arylgroups in the C2 position of each monomer, where one of these arylgroups bears a substituent designed to provide an anchor for linking thecompound to another moiety. Co-pending International applicationPCT/US2011/032664, filed 15 Apr. 2011, discloses the inclusion of thesePBD dimer compounds in targeted conjugates.

DISCLOSURE OF THE INVENTION

The present inventors have developed further specific unsymmetricaldimeric PBD compounds for inclusion in targeted conjugates. Thesecompounds may have advantages in their preparation and use, particularlyin their biological properties and the synthesis of conjugates, and thebiological properties of these conjugates.

The present invention comprises a compound, or a pharmaceuticallyacceptable salt or solvate thereof, selected from the group consistingof:

wherein either:

-   (a) R¹⁰ is H, and R¹¹ is OH or OR^(A), where R^(A) is saturated C₁₋₄    alkyl; or-   (b) R¹⁰ and R¹¹ form a nitrogen-carbon double bond between the    nitrogen and carbon atoms to which they are bound; or-   (c) R¹⁰ is H and R¹¹ is SO_(z)M, where z is 2 or 3 and M is a    monovalent pharmaceutically acceptable cation, or both M together    are a divalent pharmaceutically acceptable cation.

A second aspect of the present invention provides the use of a compoundof the first aspect of the invention in the manufacture of a medicamentfor treating a proliferative disease. The second aspect also provides acompound of the first aspect of the invention for use in the treatmentof a proliferative disease.

One of ordinary skill in the art is readily able to determine whether ornot a candidate conjugate treats a proliferative condition for anyparticular cell type. For example, assays which may conveniently be usedto assess the activity offered by a particular compound are described inthe examples below.

A third aspect of the present invention comprises a compound accordingto the first aspect of the invention, except where either:

-   (a) R¹⁰ is carbamate nitrogen protecting group, and R¹¹ is    O-Prot^(◯), wherein Prot^(◯) is an oxygen protecting group; or-   (b) R¹⁰ is a hemi-aminal nitrogen protecting group and R¹¹ is an oxo    group.

A fourth aspect of the present invention comprises a method of making acompound of the first aspect, or a pharmaceutically acceptable salt orsolvate thereof, from a compound of the third aspect, or apharmaceutically acceptable salt or solvate thereof, by deprotection ofthe imine bond.

The unsymmetrical dimeric PBD compounds of the present invention aremade by different strategies to those previously employed in makingsymmetrical dimeric PBD compounds. In particular, the present inventorshave developed a method which involves adding each each C2 substituentto a symmetrical PBD dimer core in separate method steps. Accordingly, afifth aspect of the present invention provides a method of making acompound of the first or third aspect of the invention, comprising atleast one of the method steps set out below.

In a sixth aspect, the present invention relates to Conjugatescomprising dimers of PBDs linked to a targeting agent, wherein the PBDdimer is a compound as described herein, or a pharmaceuticallyacceptable salt or solvate thereof (supra).

In some embodiments, the Conjugates have the following formula IV:

L-(LU-D)_(p)  (IV)

or a pharmaceutically acceptable salt or solvate thereof, wherein L is aLigand unit (i.e., a targeting agent), LU is a Linker unit and D is aDrug unit that is a PBD dimer (see below). The subscript p is from 1 to20. Accordingly, the Conjugates comprise a Ligand unit covalently linkedto at least one Drug unit by a Linker unit. The Ligand unit, describedmore fully below, is a targeting agent that binds to a target moiety.The Ligand unit can, for example, specifically bind to a cell component(a Cell Binding Agent) or to other target molecules of interest.Accordingly, the present invention also provides methods for thetreatment of, for example, various cancers and autoimmune disease. Thesemethods encompass the use of the Conjugates wherein the Ligand unit is atargeting agent that specifically binds to a target molecule. The Ligandunit can be, for example, a protein, polypeptide or peptide, such as anantibody, an antigen-binding fragment of an antibody, or other bindingagent, such as an Fc fusion protein.

In the conjugates of the present invention, the PBD dimer D is selectedfrom the group consisting of:

or a pharmaceutically acceptable salt or solvate thereof, where R¹⁰ andR¹¹ are as defined in the first aspect, and the asterix indicates thepoint of attachment to the Linker Unit.

The drug loading is represented by p, the number of drug molecules perLigand unit (e.g., an antibody). Drug loading may range from 1 to 20Drug units (D) per Ligand unit (e.g., Ab or mAb). For compositions, prepresents the average drug loading of the Conjugates in thecomposition, and p ranges from 1 to 20.

In some embodiments, p is from about 1 to about 8 Drug units per Ligandunit. In some embodiments, p is 1. In some embodiments, p is 2. In someembodiments, p is from about 2 to about 8 Drug units per Ligand unit. Insome embodiments, p is from about 2 to about 6, 2 to about 5, or 2 toabout 4 Drug units per Ligand unit. In some embodiments, p is about 2,about 4, about 6 or about 8 Drug units per Ligand unit.

The average number of Drugs units per Ligand unit in a preparation froma conjugation reaction may be characterized by conventional means suchas mass spectroscopy, ELISA assay, and HPLC. The quantitativedistribution of Conjugates in terms of p may also be determined. In someinstances, separation, purification, and characterization of homogeneousConjugates, where p is a certain value, from Conjugates with other drugloadings may be achieved by means such as reverse phase HPLC orelectrophoresis.

In a seventh aspect, the present invention relates to Linker-Drugcompounds (i.e., Drug-Linkers) comprising dimers of PBDs (see above)linked to a linking unit. These Drug-linkers can be used asintermediates for the synthesis of Conjugates comprising dimers of PBDslinked to a targeting agent.

These Drug-Linkers have the following formula V:

LU-D  (V)

or a pharmaceutically acceptable salt or solvate thereof, wherein LU isa Linker unit and D is a Drug unit that is a PBD dimer, as defined inthe sixth aspect of the invention.

FIGURE

FIG. 1 shows the effect on mean tumour volume following treatment withtwo conjugates of the present invention.

DEFINITIONS Pharmaceutically Acceptable Cations

Examples of pharmaceutically acceptable monovalent and divalent cationsare discussed in Berge, et al., J. Pharm. Sci., 66, 1-19 (1977), whichis incorporated herein by reference in its entirety and for allpurposes.

The pharmaceutically acceptable cation may be inorganic or organic.

Examples of pharmaceutically acceptable monovalent inorganic cationsinclude, but are not limited to, alkali metal ions such as Na⁺ and K⁺.Examples of pharmaceutically acceptable divalent inorganic cationsinclude, but are not limited to, alkaline earth cations such as Ca²⁺ andMg²⁺. Examples of pharmaceutically acceptable organic cations include,but are not limited to, ammonium ion (i.e. NH₄ ⁺) and substitutedammonium ions (e.g. NH₃R⁺, NH₂R₂ ⁺, NHR₃ ⁺, NR₄ ⁺). Examples of somesuitable substituted ammonium ions are those derived from: ethylamine,diethylamine, dicyclohexylamine, triethylamine, butylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine,phenylbenzylamine, choline, meglumine, and tromethamine, as well asamino acids, such as lysine and arginine. An example of a commonquaternary ammonium ion is N(CH₃)₄ ⁺.

Groups

The term “saturated C₁₋₄ alkyl” as used herein, pertains to a monovalentmoiety obtained by removing a hydrogen atom from a carbon atom of ahydrocarbon compound having from 1 to 4 carbon atoms, which may bealiphatic or alicyclic. Similarly, the term “saturated C₁₋₂alkyl” asused herein, pertains to a monovalent moiety obtained by removing ahydrogen atom from a carbon atom of a hydrocarbon compound having from 1to 2 carbon atoms, i.e. methyl or ethyl.

Examples of saturated alkyl groups include, but are not limited to,methyl (C₁), ethyl (C₂), propyl (C₃), and butyl (C₄)

Examples of saturated linear alkyl groups include, but are not limitedto, methyl (C₁), ethyl (C₂), n-propyl (C₃) and n-butyl (C₄).

Examples of saturated branched alkyl groups include iso-propyl (C₃),iso-butyl (C₄), sec-butyl (C₄) and tert-butyl (C₄).

Oxo (keto, -one): ═O.

Oxygen protecting group: the term “oxygen protecting group” refers to amoiety which masks a hydroxy group, and these are well known in the art.A large number of suitable groups are described on pages 23 to 200 ofGreene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis,3^(rd) Edition, John Wiley & Sons, Inc., 1999, which is incorporatedherein by reference in its entirety and for all purposes. Classes ofparticular interest include silyl ethers (e.g. TMS, TBDMS), substitutedmethyl ethers (e.g. THP) and esters (e.g. acetate).

Carbamate nitrogen protecting group: the term “carbamate nitrogenprotecting group” pertains to a moiety which masks the nitrogen in theimine bond, and these are well known in the art. These groups have thefollowing structure:

wherein R′¹⁰ is R as defined below. A large number of suitable groupsare described on pages 503 to 549 of Greene, T. W. and Wuts, G. M.,Protective Groups in Organic Synthesis, 3^(rd) Edition, John Wiley &Sons, Inc., 1999, which is incorporated herein by reference in itsentirety and for all purposes.

Hemi-aminal nitrogen protecting group: the term “hemi-aminal nitrogenprotecting group” pertains to a group having the following structure:

wherein R′¹⁰ is R as defined below. A large number of suitable groupsare described on pages 633 to 647 as amide protecting groups of Greene,T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3^(rd)Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein byreference in its entirety and for all purposes.

R

R is selected from optionally substituted C₁₋₁₂ alkyl, C₃₋₂₀heterocyclyl and C₅₋₂₀ aryl groups.

The phrase “optionally substituted” as used herein, pertains to a parentgroup which may be unsubstituted or which may be substituted.

Unless otherwise specified, the term “substituted” as used herein,pertains to a parent group which bears one or more substituents. Theterm “substituent” is used herein in the conventional sense and refersto a chemical moiety which is covalently attached to, or if appropriate,fused to, a parent group. A wide variety of substituents are well known,and methods for their formation and introduction into a variety ofparent groups are also well known.

Examples of substituents are described in more detail below.

C₁₋₁₂ alkyl: The term “C₁₋₁₂ alkyl” as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from a carbonatom of a hydrocarbon compound having from 1 to 12 carbon atoms, whichmay be aliphatic or alicyclic, and which may be saturated or unsaturated(e.g. partially unsaturated, fully unsaturated). The term “C₁₋₄ alkyl”as used herein, pertains to a monovalent moiety obtained by removing ahydrogen atom from a carbon atom of a hydrocarbon compound having from 1to 4 carbon atoms, which may be aliphatic or alicyclic, and which may besaturated or unsaturated (e.g. partially unsaturated, fullyunsaturated). Similarly, the term “C₁₋₂alkyl” as used herein, pertainsto a monovalent moiety obtained by removing a hydrogen atom from acarbon atom of a hydrocarbon compound having from 1 to 2 carbon atoms,i.e. methyl or ethyl.

Thus, the term “alkyl” includes the sub-classes alkenyl, alkynyl,cycloalkyl, etc., discussed below.

Examples of saturated alkyl groups include, but are not limited to,methyl (C₁), ethyl (C₂), propyl (C₃), butyl (C₄), pentyl (C₅), hexyl(C₆) and heptyl (C₇).

Examples of saturated linear alkyl groups include, but are not limitedto, methyl (C₁), ethyl (C₂), n-propyl (C₃), n-butyl (C₄), n-pentyl(amyl) (C₅), n-hexyl (C₆) and n-heptyl (C₇).

Examples of saturated branched alkyl groups include iso-propyl (C₃),iso-butyl (C₄), sec-butyl (C₄), tert-butyl (C₄), iso-pentyl (C₅), andneo-pentyl (C₅).

C₂₋₁₂ Alkenyl: The term “C₂₋₁₂ alkenyl” as used herein, pertains to analkyl group having one or more carbon-carbon double bonds.

Examples of unsaturated alkenyl groups include, but are not limited to,ethenyl (vinyl, —CH═CH₂), 1-propenyl (—CH═CH—CH₃), 2-propenyl (allyl,—CH—CH═CH₂), isopropenyl (1-methylvinyl, —C(CH₃)═CH₂), butenyl (C₄),pentenyl (C₅), and hexenyl (C₆).

C₂₋₁₂ alkynyl: The term “C₂₋₁₂ alkynyl” as used herein, pertains to analkyl group having one or more carbon-carbon triple bonds.

Examples of unsaturated alkynyl groups include, but are not limited to,ethynyl (—C≡CH) and 2-propynyl (propargyl, —CH₂—C≡CH).

C₃₋₁₂ cycloalkyl: The term “C₃₋₁₂ cycloalkyl” as used herein, pertainsto an alkyl group which is also a cyclyl group; that is, a monovalentmoiety obtained by removing a hydrogen atom from an alicyclic ring atomof a cyclic hydrocarbon (carbocyclic) compound, which moiety has from 3to 7 carbon atoms, including from 3 to 7 ring atoms.

Examples of cycloalkyl groups include, but are not limited to, thosederived from:

-   -   saturated monocyclic hydrocarbon compounds:        cyclopropane (C₃), cyclobutane (C₄), cyclopentane (C₅),        cyclohexane (C₆), cycloheptane (C₇), methylcyclopropane (C₄),        dimethylcyclopropane (C₅), methylcyclobutane (C₅),        dimethylcyclobutane (C₆), methylcyclopentane (C₆),        dimethylcyclopentane (C₇) and methylcyclohexane (C₇);    -   unsaturated monocyclic hydrocarbon compounds:        cyclopropene (C₃), cyclobutene (C₄), cyclopentene (C₅),        cyclohexene (C₆), methylcyclopropene (C₄), dimethylcyclopropene        (C₅), methylcyclobutene (C₅), dimethylcyclobutene (C₆),        methylcyclopentene (C₆), dimethylcyclopentene (C₇) and        methylcyclohexene (C₇); and    -   saturated polycyclic hydrocarbon compounds:        norcarane (C₇), norpinane (C₇), norbornane (C₇).

C₃₋₂₀ heterocyclyl: The term “C₃₋₂₀ heterocyclyl” as used herein,pertains to a monovalent moiety obtained by removing a hydrogen atomfrom a ring atom of a heterocyclic compound, which moiety has from 3 to20 ring atoms, of which from 1 to 10 are ring heteroatoms. Preferably,each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ringheteroatoms.

In this context, the prefixes (e.g. C₃₋₂₀, C₃₋₇, C₅₋₆, etc.) denote thenumber of ring atoms, or range of number of ring atoms, whether carbonatoms or heteroatoms. For example, the term “C₅₋₆heterocyclyl”, as usedherein, pertains to a heterocyclyl group having 5 or 6 ring atoms.

Examples of monocyclic heterocyclyl groups include, but are not limitedto, those derived from:

-   N₁: aziridine (C₃), azetidine (C₄), pyrrolidine (tetrahydropyrrole)    (C₅), pyrroline (e.g., 3-pyrroline, 2,5-dihydropyrrole) (C₅),    2H-pyrrole or 3H-pyrrole (isopyrrole, isoazole) (C₅), piperidine    (C₆), dihydropyridine (C₆), tetrahydropyridine (C₆), azepine (C₇);-   O₁: oxirane (C₃), oxetane (C₄), oxolane (tetrahydrofuran) (C₅),    oxole (dihydrofuran) (C₅), oxane (tetrahydropyran) (C₆),    dihydropyran (C₆), pyran (C₆), oxepin (C₇);-   S₁: thiirane (C₃), thietane (C₄), thiolane (tetrahydrothiophene)    (C₅), thiane (tetrahydrothiopyran) (C₆), thiepane (C₇);-   O₂: dioxolane (C₅), dioxane (C₆), and dioxepane (C₇);-   O₃: trioxane (C₆);-   N₂: imidazolidine (C₅), pyrazolidine (diazolidine) (C₅), imidazoline    (C₅), pyrazoline (dihydropyrazole) (C₅), piperazine (C₆);-   N₁O₁: tetrahydrooxazole (C₅), dihydrooxazole (C₅),    tetrahydroisoxazole (C₅), dihydroisoxazole (C₅), morpholine (C₆),    tetrahydrooxazine (C₆), dihydrooxazine (C₆), oxazine (C₆);-   N₁S₁: thiazoline (C₅), thiazolidine (C₅), thiomorpholine (C₆); N₂O₁:    oxadiazine (C₆);-   O₁S₁: oxathiole (C₅) and oxathiane (thioxane) (C₆); and, N₁O₁S₁:    oxathiazine (C₆).

Examples of substituted monocyclic heterocyclyl groups include thosederived from saccharides, in cyclic form, for example, furanoses (C₅),such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse,and pyranoses (C₆), such as allopyranose, altropyranose, glucopyranose,mannopyranose, gulopyranose, idopyranose, galactopyranose, andtalopyranose.

C₅₋₂₀ aryl: The term “C₅₋₂₀ aryl”, as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from an aromaticring atom of an aromatic compound, which moiety has from 3 to 20 ringatoms. The term “C₅₋₇ aryl”, as used herein, pertains to a monovalentmoiety obtained by removing a hydrogen atom from an aromatic ring atomof an aromatic compound, which moiety has from 5 to 7 ring atoms and theterm “C₅₋₁₀ aryl”, as used herein, pertains to a monovalent moietyobtained by removing a hydrogen atom from an aromatic ring atom of anaromatic compound, which moiety has from 5 to 10 ring atoms. Preferably,each ring has from 5 to 7 ring atoms.

In this context, the prefixes (e.g. C₃₋₂₀, C₅₋₇, C₅₋₆, C₅₋₁₀, etc.)denote the number of ring atoms, or range of number of ring atoms,whether carbon atoms or heteroatoms. For example, the term “C₅₋₆ aryl”as used herein, pertains to an aryl group having 5 or 6 ring atoms.

The ring atoms may be all carbon atoms, as in “carboaryl groups”.

Examples of carboaryl groups include, but are not limited to, thosederived from benzene (i.e. phenyl) (C₆), naphthalene (C₁₀), azulene(C₁₀), anthracene (C₁₄), phenanthrene (C₁₄), naphthacene (C₁₈), andpyrene (C₁₆).

Examples of aryl groups which comprise fused rings, at least one ofwhich is an aromatic ring, include, but are not limited to, groupsderived from indane (e.g. 2,3-dihydro-1H-indene) (C₉), indene (C₉),isoindene (C₉), tetraline (1,2,3,4-tetrahydronaphthalene (C₁₀),acenaphthene (C₁₂), fluorene (C₁₃), phenalene (C₁₃), acephenanthrene(C₁₅), and aceanthrene (C₁₆).

Alternatively, the ring atoms may include one or more heteroatoms, as in“heteroaryl groups”. Examples of monocyclic heteroaryl groups include,but are not limited to, those derived from:

-   N₁: pyrrole (azole) (C₅), pyridine (azine) (C₆);-   O₁: furan (oxole) (C₅);-   S₁: thiophene (thiole) (C₅);-   N₁O₁: oxazole (C₅), isoxazole (C₅), isoxazine (C₆);-   N₂O₁: oxadiazole (furazan) (C₅);-   N₃O₁: oxatriazole (C₅);-   N₁S₁: thiazole (C₅), isothiazole (C₅);-   N₂: imidazole (1,3-diazole) (C₅), pyrazole (1,2-diazole) (C₅),    pyridazine (1,2-diazine) (C₆), pyrimidine (1,3-diazine) (C₆) (e.g.,    cytosine, thymine, uracil), pyrazine (1,4-diazine) (C₆);-   N₃: triazole (C₅), triazine (C₆); and,-   N₄: tetrazole (C₅).

Examples of heteroaryl which comprise fused rings, include, but are notlimited to:

-   -   C₉ (with 2 fused rings) derived from benzofuran (O₁),        isobenzofuran (O₁), indole (N₁), isoindole (N₁), indolizine        (N₁), indoline (N₁), isoindoline (N₁), purine (N₄) (e.g.,        adenine, guanine), benzimidazole (N₂), indazole (N₂),        benzoxazole (N₁O₁), benzisoxazole (N₁O₁), benzodioxole (O₂),        benzofurazan (N₂O₁), benzotriazole (N₃), benzothiofuran (S₁),        benzothiazole (N₁S₁), benzothiadiazole (N₂S);    -   C₁₀ (with 2 fused rings) derived from chromene (O₁), isochromene        (O₁), chroman (O₁), isochroman (O₁), benzodioxan (O₂), quinoline        (N₁), isoquinoline (N₁), quinolizine (N₁), benzoxazine (N₁O₁),        benzodiazine (N₂), pyridopyridine (N₂), quinoxaline (N₂),        quinazoline (N₂), cinnoline (N₂), phthalazine (N₂),        naphthyridine (N₂), pteridine (N₄);    -   C₁₁ (with 2 fused rings) derived from benzodiazepine (N₂);    -   C₁₃ (with 3 fused rings) derived from carbazole (N₁),        dibenzofuran (O₁), dibenzothiophene (S₁), carboline (N₂),        perimidine (N₂), pyridoindole (N₂); and,    -   C₁₄ (with 3 fused rings) derived from acridine (N₁), xanthene        (O₁), thioxanthene (S₁), oxanthrene (O₂), phenoxathiin (O₁S₁),        phenazine (N₂), phenoxazine (N₁O₁), phenothiazine (N₁S₁),        thianthrene (S₂), phenanthridine (N₁), phenanthroline (N₂),        phenazine (N₂).

The above groups, whether alone or part of another substituent, maythemselves optionally be substituted with one or more groups selectedfrom themselves and the additional substituents listed below.

Halo: —F, —Cl, —Br, and —I.

Hydroxy: —OH.

Ether: —OR, wherein R is an ether substituent, for example, a C₁₋₇ alkylgroup (also referred to as a C₁₋₇ alkoxy group, discussed below), aC₃₋₂₀ heterocyclyl group (also referred to as a C₃₋₂₀ heterocyclyloxygroup), or a C₅₋₂₀ aryl group (also referred to as a C₅₋₂₀ aryloxygroup), preferably a C₁₋₇alkyl group.

Alkoxy: —OR, wherein R is an alkyl group, for example, a C₁₋₇ alkylgroup. Examples of C₁₋₇ alkoxy groups include, but are not limited to,—OMe (methoxy), —OEt (ethoxy), —O(nPr) (n-propoxy), —O(iPr)(isopropoxy), —O(nBu) (n-butoxy), —O(sBu) (sec-butoxy), —O(iBu)(isobutoxy), and —O(tBu) (tert-butoxy).

Acetal: —CH(OR¹)(OR²), wherein R¹ and R² are independently acetalsubstituents, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group, or, in thecase of a “cyclic” acetal group, R¹ and R², taken together with the twooxygen atoms to which they are attached, and the carbon atoms to whichthey are attached, form a heterocyclic ring having from 4 to 8 ringatoms. Examples of acetal groups include, but are not limited to,—CH(OMe)₂, —CH(OEt)₂, and —CH(OMe)(OEt).

Hemiacetal: —CH(OH)(OR¹), wherein R¹ is a hemiacetal substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group.

Examples of hemiacetal groups include, but are not limited to,—CH(OH)(OMe) and —CH(OH)(OEt).

Ketal: —CR(OR¹)(OR²), where R¹ and R² are as defined for acetals, and Ris a ketal substituent other than hydrogen, for example, a C₁₋₇ alkylgroup, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably aC₁₋₇ alkyl group. Examples ketal groups include, but are not limited to,—C(Me)(OMe)₂, —C(Me)(OEt)₂, —C(Me)(OMe)(OEt), —C(Et)(OMe)₂,—C(Et)(OEt)₂, and —C(Et)(OMe)(OEt).

Hemiketal: —CR(OH)(OR¹), where R¹ is as defined for hemiacetals, and Ris a hemiketal substituent other than hydrogen, for example, a C₁₋₇alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group. Examples of hemiacetal groups include,but are not limited to, —C(Me)(OH)(OMe), —C(Et)(OH)(OMe),—C(Me)(OH)(OEt), and —C(Et)(OH) (OEt).

Oxo (keto, -one): ═O.

Thione (thioketone): ═S.

Imino (imine): ═NR, wherein R is an imino substituent, for example,hydrogen, C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably hydrogen or a C₁₋₇ alkyl group. Examples of estergroups include, but are not limited to, ═NH, ═NMe, ═NEt, and ═NPh.

Formyl (carbaldehyde, carboxaldehyde): —C(═O)H.

Acyl (keto): —C(═O)R, wherein R is an acyl substituent, for example, aC₁₋₇ alkyl group (also referred to as C₁₋₇alkylacyl or C₁₋₇alkanoyl), aC₃₋₂₀ heterocyclyl group (also referred to as C₃₋₂₀ heterocyclylacyl),or a C₅₋₂₀ aryl group (also referred to as C₅₋₂₀ arylacyl), preferably aC₁₋₇ alkyl group. Examples of acyl groups include, but are not limitedto, —C(═O)CH₃ (acetyl), —C(═O)CH₂CH₃ (propionyl), —C(═O)C(CH₃)₃(t-butyryl), and —C(═O)Ph (benzoyl, phenone).

Carboxy (carboxylic acid): —C(═O)OH.

Thiocarboxy (thiocarboxylic acid): —C(═S)SH.

Thiolocarboxy (thiolocarboxylic acid): —C(═O)SH.

Thionocarboxy (thionocarboxylic acid): —C(═S)OH.

Imidic acid: —C(═NH)OH.

Hydroxamic acid: —C(═NOH)OH.

Ester (carbon/late, carboxylic acid ester, oxycarbonyl): —C(═O)OR,wherein R is an ester substituent, for example, a C₁₋₇ alkyl group, aC₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkylgroup. Examples of ester groups include, but are not limited to,—C(═O)OCH₃, —C(═O)OCH₂CH₃, —C(═O)OC(CH₃)₃, and —C(═O)OPh.

Acyloxy (reverse ester): —OC(═O)R, wherein R is an acyloxy substituent,for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀aryl group, preferably a C₁₋₇ alkyl group. Examples of acyloxy groupsinclude, but are not limited to, —OC(═O)CH₃ (acetoxy), —OC(═O)CH₂CH₃,—OC(═O)C(CH₃)₃, —OC(═O)Ph, and —OC(═O)CH₂Ph.

Oxycarboyloxy: —OC(═O)OR, wherein R is an ester substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group. Examples of ester groups include,but are not limited to, —OC(═O)OCH₃, —OC(═O)OCH₂CH₃, —OC(═O)OC(CH₃)₃,and —OC(═O)OPh.

Amino: —NR¹R², wherein R¹ and R² are independently amino substituents,for example, hydrogen, a C₁₋₇ alkyl group (also referred to as C₁₋₇alkylamino or di-C₁₋₇ alkylamino), a C₃₋₂₀ heterocyclyl group, or aC₅₋₂₀ aryl group, preferably H or a C₁₋₇ alkyl group, or, in the case ofa “cyclic” amino group, R¹ and R², taken together with the nitrogen atomto which they are attached, form a heterocyclic ring having from 4 to 8ring atoms. Amino groups may be primary (—NH₂), secondary (—NHR¹), ortertiary (—NHR¹R²), and in cationic form, may be quaternary (−⁺NR¹R²R³).Examples of amino groups include, but are not limited to, —NH₂, —NHCH₃,—NHC(CH₃)₂, —N(CH₃)₂, —N(CH₂CH₃)₂, and —NHPh. Examples of cyclic aminogroups include, but are not limited to, aziridino, azetidino,pyrrolidino, piperidino, piperazino, morpholino, and thiomorpholino.

Amido (carbamoyl, carbamyl, aminocarbonyl, carboxamide): —C(═O)NR¹R²,wherein R¹ and R² are independently amino substituents, as defined foramino groups. Examples of amido groups include, but are not limited to,—C(═O)NH₂, —C(═O)NHCH₃, —C(═O)N(CH₃)₂, —C(═O)NHCH₂CH₃, and—C(═O)N(CH₂CH₃)₂, as well as amido groups in which R¹ and R², togetherwith the nitrogen atom to which they are attached, form a heterocyclicstructure as in, for example, piperidinocarbonyl, morpholinocarbonyl,thiomorpholinocarbonyl, and piperazinocarbonyl.

Thioamido (thiocarbamyl): —C(═S)NR¹R², wherein R¹ and R² areindependently amino substituents, as defined for amino groups. Examplesof amido groups include, but are not limited to, —C(═S)NH₂, —C(═S)NHCH₃,—C(═S)N(CH₃)₂, and —C(═S)NHCH₂CH₃.

Acylamido (acylamino): —NR¹C(═O)R², wherein R¹ is an amide substituent,for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group,or a C₅₋₂₀ aryl group, preferably hydrogen or a C₁₋₇ alkyl group, and R²is an acyl substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀heterocyclyl group, or a C₅₋₂₀aryl group, preferably hydrogen or a C₁₋₇alkyl group. Examples of acylamide groups include, but are not limitedto, —NHC(═O)CH₃, —NHC(═O)CH₂CH₃, and —NHC(═O)Ph. R¹ and R² may togetherform a cyclic structure, as in, for example, succinimidyl, maleimidyl,and phthalimidyl:

Aminocarbonyloxy: —OC(═O)NR¹R², wherein R¹ and R² are independentlyamino substituents, as defined for amino groups. Examples ofaminocarbonyloxy groups include, but are not limited to, —OC(═O)NH₂,—OC(═O)NHMe, —OC(═O)NMe₂, and —OC(═O)NEt₂.

Ureido: —N(R¹)CONR²R³ wherein R² and R³ are independently aminosubstituents, as defined for amino groups, and R¹ is a ureidosubstituent, for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀heterocyclyl group, or a C₅₋₂₀ aryl group, preferably hydrogen or a C₁₋₇alkyl group. Examples of ureido groups include, but are not limited to,—NHCONH₂, —NHCONHMe, —NHCONHEt, —NHCONMe₂, —NHCONEt₂, —NMeCONH₂,—NMeCONHMe, —NMeCONHEt, —NMeCONMe₂, and —NMeCONEt₂.

Guanidino: —NH—C(═NH)NH₂.

Tetrazolyl: a five membered aromatic ring having four nitrogen atoms andone carbon atom,

Imino: ═NR, wherein R is an imino substituent, for example, for example,hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀aryl group, preferably H or a C₁₋₇alkyl group. Examples of imino groupsinclude, but are not limited to, ═NH, ═NMe, and =NEt.

Amidine (amidino): —C(═NR)NR₂, wherein each R is an amidine substituent,for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group,or a C₅₋₂₀ aryl group, preferably H or a C₁₋₇ alkyl group. Examples ofamidine groups include, but are not limited to, —C(═NH)NH₂, —C(═NH)NMe₂,and —C(═NMe)NMe₂.

Nitro: —NO₂.

Nitroso: —NO.

Azido: —N₃.

Cyano (nitrile, carbonitrile): —CN.

Isocyano: —NC.

Cyanato: —OCN.

Isocyanato: —NCO.

Thiocyano (thiocyanato): —SCN.

Isothiocyano (isothiocyanato): —NCS.

Sulfhydryl (thiol, mercapto): —SH.

Thioether (sulfide): —SR, wherein R is a thioether substituent, forexample, a C₁₋₇ alkyl group (also referred to as a C₁₋₇alkylthio group),a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇alkyl group. Examples of C₁₋₇alkylthio groups include, but are notlimited to, —SCH₃ and —SCH₂CH₃.

Disulfide: —SS—R, wherein R is a disulfide substituent, for example, aC₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group (also referred to herein as C₁₋₇ alkyldisulfide). Examples of C₁₋₇ alkyl disulfide groups include, but are notlimited to, —SSCH₃ and —SSCH₂CH₃.

Sulfine (sulfinyl, sulfoxide): —S(═O)R, wherein R is a sulfinesubstituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples ofsulfine groups include, but are not limited to, —S(═O)CH₃ and—S(═O)CH₂CH₃.

Sulfone (sulfonyl): —S(═O)₂R, wherein R is a sulfone substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group, including, for example, afluorinated or perfluorinated C₁₋₇ alkyl group. Examples of sulfonegroups include, but are not limited to, —S(═O)₂CH₃ (methanesulfonyl,mesyl), —S(═O)₂CF₃ (triflyl), —S(═O)₂CH₂CH₃ (esyl), —S(═O)₂C₄F₉(nonaflyl), —S(═O)₂CH₂CF₃ (tresyl), —S(═O)₂CH₂CH₂NH₂ (tauryl), —S(═O)₂Ph(phenylsulfonyl, besyl), 4-methylphenylsulfonyl (tosyl),4-chlorophenylsulfonyl (closyl), 4-bromophenylsulfonyl (brosyl),4-nitrophenyl (nosyl), 2-naphthalenesulfonate (napsyl), and5-dimethylamino-naphthalen-1-ylsulfonate (dansyl).

Sulfinic acid (sulfino): —S(═O)OH, —SO₂H.

Sulfonic acid (sulfo): —S(═O)₂OH, —SO₃H.

Sulfinate (sulfinic acid ester): —S(═O)OR; wherein R is a sulfinatesubstituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples ofsulfinate groups include, but are not limited to, —S(═O)OCH₃(methoxysulfinyl; methyl sulfinate) and —S(═O)OCH₂CH₃ (ethoxysulfinyl;ethyl sulfinate).

Sulfonate (sulfonic acid ester): —S(═O)₂OR, wherein R is a sulfonatesubstituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples ofsulfonate groups include, but are not limited to, —S(═O)₂OCH₃(methoxysulfonyl; methyl sulfonate) and —S(═O)₂OCH₂CH₃ (ethoxysulfonyl;ethyl sulfonate).

Sulfinyloxy: —OS(═O)R, wherein R is a sulfinyloxy substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group.

Examples of sulfinyloxy groups include, but are not limited to,—OS(═O)CH₃ and —OS(═O)CH₂CH₃.

Sulfonyloxy: —OS(═O)₂R, wherein R is a sulfonyloxy substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group. Examples of sulfonyloxy groupsinclude, but are not limited to, —OS(═O)₂CH₃ (mesylate) and—OS(═O)₂CH₂CH₃ (esylate).

Sulfate: —OS(═O)₂OR; wherein R is a sulfate substituent, for example, aC₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group. Examples of sulfate groups include, butare not limited to, —OS(═O)₂OCH₃ and —SO(═O)₂OCH₂CH₃.

Sulfamyl (sulfamoyl; sulfinic acid amide; sulfinamide): —S(═O)NR¹R²,wherein R¹ and R² are independently amino substituents, as defined foramino groups. Examples of sulfamyl groups include, but are not limitedto, —S(═O)NH₂, —S(═O)NH(CH₃), —S(═O)N(CH₃)₂, —S(═O)NH(CH₂CH₃),—S(═O)N(CH₂CH₃)₂, and —S(═O)NHPh.

Sulfonamido (sulfinamoyl; sulfonic acid amide; sulfonamide):—S(═O)₂NR¹R², wherein R¹ and R² are independently amino substituents, asdefined for amino groups. Examples of sulfonamido groups include, butare not limited to, —S(═O)₂NH₂, —S(═O)₂NH(CH₃), —S(═O)₂N(CH₃)₂,—S(═O)₂NH(CH₂CH₃), —S(═O)₂N(CH₂CH₃)₂, and —S(═O)₂NHPh.

Sulfamino: —NR¹S(═O)₂OH, wherein R¹ is an amino substituent, as definedfor amino groups. Examples of sulfamino groups include, but are notlimited to, —NHS(═O)₂OH and —N(CH₃)S(═O)₂OH.

Sulfonamino: —NR¹S(═O)₂R, wherein R¹ is an amino substituent, as definedfor amino groups, and R is a sulfonamino substituent, for example, aC₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group. Examples of sulfonamino groups include,but are not limited to, —NHS(═O)₂CH₃ and —N(CH₃)S(═O)₂C₆H₅.

Sulfinamino: —NR¹S(═O)R, wherein R¹ is an amino substituent, as definedfor amino groups, and R is a sulfinamino substituent, for example, aC₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group. Examples of sulfinamino groups include,but are not limited to, —NHS(═O)CH₃ and —N(CH₃)S(═O)C₆H₅.

Phosphino (phosphine): —PR₂, wherein R is a phosphino substituent, forexample, —H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀aryl group, preferably —H, a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group.Examples of phosphino groups include, but are not limited to, —PH₂,—P(CH₃)₂, —P(CH₂CH₃)₂, —P(t-Bu)₂, and —P(Ph)₂.

Phospho: —P(═O)₂.

Phosphinyl (phosphine oxide): —P(═O)R₂, wherein R is a phosphinylsubstituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group or a C₅₋₂₀aryl group. Examples of phosphinyl groups include, but are not limitedto, —P(═O)(CH₃)₂, —P(═O)(CH₂CH₃)₂, —P(═O)(t-Bu)₂, and —P(═O)(Ph)₂.

Phosphonic acid (phosphono): —P(═O)(OH)₂.

Phosphonate (phosphono ester): —P(═O)(OR)₂, where R is a phosphonatesubstituent, for example, —H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably —H, a C₁₋₇ alkyl group, or aC₅₋₂₀ aryl group. Examples of phosphonate groups include, but are notlimited to, —P(═O)(OCH₃)₂, —P(═O)(OCH₂CH₃)₂, —P(═O)(O-t-Bu)₂, and—P(═O)(OPh)₂.

Phosphoric acid (phosphonooxy): —OP(═O)(OH)₂.

Phosphate (phosphonooxy ester): —OP(═O)(OR)₂, where R is a phosphatesubstituent, for example, —H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably —H, a C₁₋₇ alkyl group, or aC₅₋₂₀ aryl group. Examples of phosphate groups include, but are notlimited to, —OP(═O)(OCH₃)₂, —OP(═O)(OCH₂CH₃)₂, —OP(═O)(O-t-Bu)₂, and—OP(═O)(OPh)₂.

Phosphorous acid: —OP(OH)₂.

Phosphite: —OP(OR)₂, where R is a phosphite substituent, for example,—H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably —H, a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group.Examples of phosphite groups include, but are not limited to,—OP(OCH₃)₂, —OP(OCH₂CH₃)₂, —OP(O-t-Bu)₂, and —OP(OPh)₂.

Phosphoramidite: —OP(OR¹)—NR² ₂, where R¹ and R² are phosphoramiditesubstituents, for example, —H, a (optionally substituted) C₁₋₇ alkylgroup, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably —H,a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group. Examples of phosphoramiditegroups include, but are not limited to, —OP(OCH₂CH₃)—N(CH₃)₂,—OP(OCH₂CH₃)—N(i-Pr)₂, and —OP(OCH₂CH₂CN)—N(i-Pr)₂.

Phosphoramidate: —OP(═O)(OR¹)—NR² ₂, where R¹ and R² are phosphoramidatesubstituents, for example, —H, a (optionally substituted) C₁₋₇ alkylgroup, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably —H,a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group.

Examples of phosphoramidate groups include, but are not limited to,—OP(═O)(OCH₂CH₃)—N(CH₃)₂, —OP(═O)(OCH₂CH₃)—N(i-Pr)₂, and—OP(═O)(OCH₂CH₂CN)—N(i-Pr)₂.

Conjugates

The present invention provides Conjugates comprising a PBD dimerconnected to a Ligand unit via a Linker unit. In one embodiment, theLinker unit includes a Stretcher unit (A), a Specificity unit (L¹), anda Spacer unit (L²). The Linker unit is connected at one end to theLigand unit (L) and at the other end to the PBD dimer compound (D).

In one aspect, such a Conjugate is shown below in formula IVa:

L-(A¹ _(a)-L¹ _(s)-L² _(y)-D)_(p)  (IVa)

-   -   or a pharmaceutically acceptable salt or solvate thereof,        wherein:    -   L is the Ligand unit; and    -   -A¹ _(a)-L¹ _(s)-L² _(y)- is a Linker unit (LU), wherein:    -   -A¹- is a Stretcher unit,    -   a is 1 or 2,    -   -L¹- is a Specificity unit,    -   s is an integer ranging from 0 to 12,    -   -L²- is a Spacer unit,    -   y is 0, 1 or 2;    -   -D is a PBD dimer; and    -   p is from 1 to 20.

In another aspect, such a Conjugate is shown below in formula IVb:

Also illustrated as:

L-(A¹ _(a)-L² _(y)(-L¹ _(s))-D)_(p)  (IVb)

-   -   or a pharmaceutically acceptable salt or solvate thereof,        wherein:    -   L is the Ligand unit; and    -   -A¹ _(a)-L¹ _(s)(L² _(y))- is a Linker unit (LU), wherein:    -   -A¹- is a Stretcher unit linked to a Stretcher unit (L²),    -   a is 1 or 2,    -   -L¹- is a Specificity unit linked to a Stretcher unit (L²),    -   s is an integer ranging from 0 to 12,    -   -L²- is a Spacer unit,    -   y is 0, 1 or 2;    -   -D is a PBD dimer; and    -   p is from 1 to 20.

Preferences

The following preferences may apply to all aspects of the invention asdescribed above, or may relate to a single aspect. The preferences maybe combined together in any combination.

In one embodiment, the Conjugate has the formula:

L-(A¹ _(a)-L¹ _(s)-L² _(y)-D)_(p)

L-(A¹ _(a)-L_(s) ¹-D)_(p)

L-(A¹-L¹-D)_(p), or

L-(A¹-D)_(p)

-   -   or a pharmaceutically acceptable salt or solvate thereof,        wherein L, A¹, a, L¹ s, L², D, y and p are as described above.

In one embodiment, the Ligand unit (L) is a Cell Binding Agent (CBA)that specifically binds to a target molecule on the surface of a targetcell. An exemplary formula is illustrated below:

-   -   where the asterisk indicates the point of attachment to the Drug        unit (D), CBA is the Cell Binding Agent, L¹ is a Specificity        unit, A¹ is a Stretcher unit connecting L¹ to the Cell Binding        Agent, L² is a Spacer unit, which is a covalent bond, a        self-immolative group or together with —OC(═O)— forms a        self-immolative group, and L² is optional. —OC(═O)— may be        considered as being part of L¹ or L², as appropriate.

In another embodiment, the Ligand unit (L) is a Cell Binding Agent (CBA)that specifically binds to a target molecule on the surface of a targetcell. An exemplary formula is illustrated below:

CBA-A¹ _(a)-L¹ _(s)-L² _(y)-*

-   -   where the asterisk indicates the point of attachment to the Drug        unit (D), CBA is the Cell Binding Agent, L¹ is a Specificity        unit, A¹ is a Stretcher unit connecting L¹ to the Cell Binding        Agent, L² is a Spacer unit which is a covalent bond or a        self-immolative group, and a is 1 or 2, s is 0, 1 or 2, and y is        0 or 1 or 2.

In the embodiments illustrated above, L¹ can be a cleavable Specificityunit, and may be referred to as a “trigger” that when cleaved activatesa self-immolative group (or self-immolative groups) L², when aself-immolative group(s) is present. When the Specificity unit L¹ iscleaved, or the linkage (i.e., the covalent bond) between L¹ and L² iscleaved, the self-immolative group releases the Drug unit (D).

In another embodiment, the Ligand unit (L) is a Cell Binding Agent (CBA)that specifically binds to a target molecule on the surface of a targetcell. An exemplary formula is illustrated below:

-   -   where the asterisk indicates the point of attachment to the Drug        (D), CBA is the Cell Binding Agent, L¹ is a Specificity unit        connected to L², A¹ is a Stretcher unit connecting L² to the        Cell Binding Agent, L² is a self-immolative group, and a is 1 or        2, s is 1 or 2, and y is 1 or 2.

In the various embodiments discussed herein, the nature of L¹ and L² canvary widely. These groups are chosen on the basis of theircharacteristics, which may be dictated in part, by the conditions at thesite to which the conjugate is delivered. Where the Specificity unit L¹is cleavable, the structure and/or sequence of L¹ is selected such thatit is cleaved by the action of enzymes present at the target site (e.g.,the target cell). L¹ units that are cleavable by changes in pH (e.g.acid or base labile), temperature or upon irradiation (e.g. photolabile)may also be used. L¹ units that are cleavable under reducing oroxidising conditions may also find use in the Conjugates.

In some embodiments, L¹ may comprise one amino acid or a contiguoussequence of amino acids. The amino acid sequence may be the targetsubstrate for an enzyme.

In one embodiment, L¹ is cleavable by the action of an enzyme. In oneembodiment, the enzyme is an esterase or a peptidase. For example, L¹may be cleaved by a lysosomal protease, such as a cathepsin.

In one embodiment, L² is present and together with —C(═O)O— forms aself-immolative group or self-immolative groups. In some embodiments,—C(═O)O— also is a self-immolative group.

In one embodiment, where L¹ is cleavable by the action of an enzyme andL² is present, the enzyme cleaves the bond between L¹ and L², wherebythe self-immolative group(s) release the Drug unit.

L¹ and L², where present, may be connected by a bond selected from:

-   -   —C(═O)NH—,    -   —C(═O)O—,    -   —NHC(═O)—,    -   —OC(═O)—,    -   —OC(═O)O—,    -   —NHC(═O)O—,    -   —OC(═O)NH—,    -   —NHC(═O)NH, and    -   —O— (a glycosidic bond).

An amino group of L¹ that connects to L² may be the N-terminus of anamino acid or may be derived from an amino group of an amino acid sidechain, for example a lysine amino acid side chain.

A carboxyl group of L¹ that connects to L² may be the C-terminus of anamino acid or may be derived from a carboxyl group of an amino acid sidechain, for example a glutamic acid amino acid side chain.

A hydroxy group of L¹ that connects to L² may be derived from a hydroxygroup of an amino acid side chain, for example a serine amino acid sidechain.

In one embodiment, —C(═O)O— and L² together form the group:

-   -   where the asterisk indicates the point of attachment to the Drug        unit, the wavy line indicates the point of attachment to the L¹,        Y is —N(H)—, —O—, —C(═O)N(H)— or —C(═O)O—, and n is 0 to 3. The        phenylene ring is optionally substituted with one, two or three        substituents as described herein.

In one embodiment, Y is NH.

In one embodiment, n is 0 or 1. Preferably, n is 0.

Where Y is NH and n is 0, the self-immolative group may be referred toas a p-aminobenzylcarbonyl linker (PABC).

The self-immolative group will allow for release of the Drug unit (i.e.,the asymmetric PBD) when a remote site in the linker is activated,proceeding along the lines shown below (for n=0):

-   -   where the asterisk indicates the attachment to the Drug, L* is        the activated form of the remaining portion of the linker and        the released Drug unit is not shown. These groups have the        advantage of separating the site of activation from the Drug.

In another embodiment, —C(═O)O— and L² together form a group selectedfrom:

-   -   where the asterisk, the wavy line, Y, and n are as defined        above. Each phenylene ring is optionally substituted with one,        two or three substituents as described herein. In one        embodiment, the phenylene ring having the Y substituent is        optionally substituted and the phenylene ring not having the Y        substituent is unsubstituted.

In another embodiment, —C(═O)O— and L² together form a group selectedfrom:

-   -   where the asterisk, the wavy line, Y, and n are as defined        above, E is O, S or NR, D is N, CH, or CR, and F is N, CH, or        CR.

In one embodiment, D is N.

In one embodiment, D is CH.

In one embodiment, E is O or S.

In one embodiment, F is CH.

In a preferred embodiment, the covalent bond between L¹ and L² is acathepsin labile (e.g., cleavable) bond.

In one embodiment, L¹ comprises a dipeptide. The amino acids in thedipeptide may be any combination of natural amino acids and non-naturalamino acids. In some embodiments, the dipeptide comprises natural aminoacids. Where the linker is a cathepsin labile linker, the dipeptide isthe site of action for cathepsin-mediated cleavage. The dipeptide thenis a recognition site for cathepsin.

In one embodiment, the group —X₁—X₂— in dipeptide, —NH—X₁—X₂—CO—, isselected from:

-   -   -Phe-Lys-,    -   -Val-Ala-,    -   -Val-Lys-,    -   -Ala-Lys-,    -   -Val-Cit-,    -   -Phe-Cit-,    -   -Leu-Cit-,    -   -Ile-Cit-,    -   -Phe-Arg-, and    -   -Trp-Cit-;        where Cit is citrulline. In such a dipeptide, —NH— is the amino        group of X₁, and CO is the carbonyl group of X₂.

Preferably, the group —X₁—X₂— in dipeptide, —NH—X₁—X₂—CO—, is selectedfrom:

-   -   -Phe-Lys-,    -   -Val-Ala-,    -   -Val-Lys-,    -   -Ala-Lys-, and    -   -Val-Cit-.

Most preferably, the group —X₁—X₂— in dipeptide, —NH—X₁—X₂—CO—, is-Phe-Lys-, Val-Cit or -Val-Ala-.

Other dipeptide combinations of interest include:

-   -   -Gly-Gly-,    -   -Pro-Pro-, and    -   -Val-Glu-.

Other dipeptide combinations may be used, including those described byDubowchik et al., which is incorporated herein by reference in itsentirety and for all purposes.

In one embodiment, the amino acid side chain is chemically protected,where appropriate. The side chain protecting group may be a group asdiscussed below. Protected amino acid sequences are cleavable byenzymes. For example, a dipeptide sequence comprising a Boc sidechain-protected Lys residue is cleavable by cathepsin.

Protecting groups for the side chains of amino acids are well known inthe art and are described in the Novabiochem Catalog. Additionalprotecting group strategies are set out in Protective groups in OrganicSynthesis, Greene and Wuts.

Possible side chain protecting groups are shown below for those aminoacids having reactive side chain functionality:

-   -   Arg: Z, Mtr, Tos;    -   Asn: Trt, Xan;    -   Asp: Bzl, t-Bu;    -   Cys: Acm, Bzl, Bzl-OMe, Bzl-Me, Trt;    -   Glu: Bzl, t-Bu;    -   Gln: Trt, Xan;    -   His: Boc, Dnp, Tos, Trt;    -   Lys: Boc, Z—Cl, Fmoc, Z;    -   Ser: Bzl, TBDMS, TBDPS;    -   Thr: Bz;    -   Trp: Boc;    -   Tyr: Bzl, Z, Z—Br.

In one embodiment, —X₂— is connected indirectly to the Drug unit. Insuch an embodiment, the Spacer unit L² is present.

In one embodiment, —X₂— is connected directly to the Drug unit. In suchan embodiment, the Spacer unit L² is absent.

In one embodiment, the dipeptide is used in combination with aself-immolative group(s) (the Spacer unit). The self-immolative group(s)may be connected to —X₂—.

Where a self-immolative group is present, —X₂— is connected directly tothe self-immolative group. In one embodiment, —X₂— is connected to thegroup Y of the self-immolative group. Preferably the group —X₂—CO— isconnected to Y, where Y is NH.

In one embodiment, —X₁— is connected directly to A¹. Preferably thegroup NH—X₁— (the amino terminus of X₁) is connected to A¹. A¹ maycomprise the functionality —CO— thereby to form an amide link with —X₁—.

In one embodiment, L¹ and L² together with —OC(═O)— comprise the group—X₁—X₂-PABC—. The PABC group is connected directly to the Drug unit. Inone example, the self-immolative group and the dipeptide together formthe group -Phe-Lys-PABC-, which is illustrated below:

-   -   where the asterisk indicates the point of attachment to the Drug        unit, and the wavy line indicates the point of attachment to the        remaining portion of L¹ or the point of attachment to A¹.        Preferably, the wavy line indicates the point of attachment to        A¹.

Alternatively, the self-immolative group and the dipeptide together formthe group -Val-Ala-PABC-, which is illustrated below:

-   -   where the asterisk and the wavy line are as defined above.

In another embodiment, L¹ and L² together with —OC(═O)— represent:

-   -   where the asterisk indicates the point of attachment to the Drug        unit, the wavy line indicates the point of attachment to A¹, Y        is a covalent bond or a functional group, and E is a group that        is susceptible to cleavage thereby to activate a self-immolative        group.

E is selected such that the group is susceptible to cleavage, e.g., bylight or by the action of an enzyme. E may be —NO₂ or glucuronic acid(e.g., β-glucuronic acid). The former may be susceptible to the actionof a nitroreductase, the latter to the action of a β-glucuronidase.

The group Y may be a covalent bond.

The group Y may be a functional group selected from:

-   -   —C(═O)—    -   —NH—    -   —O—    -   —C(═O)NH—,    -   —C(═O)O—,    -   —NHC(═O)—,    -   —OC(═O)—,    -   —OC(═O)O—,    -   —NHC(═O)O—,    -   —OC(═O)NH—,    -   —NHC(═O)NH—,    -   —NHC(═O)NH,    -   —C(═O)NHC(═O)—,    -   SO₂, and    -   —S—.

The group Y is preferably —NH—, —CH₂—, —O—, and —S—.

In some embodiments, L¹ and L² together with —OC(═O)— represent:

-   -   where the asterisk indicates the point of attachment to the Drug        unit, the wavy line indicates the point of attachment to A, Y is        a covalent bond or a functional group and E is glucuronic acid        (e.g., β-glucuronic acid). Y is preferably a functional group        selected from —NH—.

In some embodiments, L¹ and L² together represent:

-   -   where the asterisk indicates the point of attachment to the        remainder of L² or the Drug unit, the wavy line indicates the        point of attachment to A¹, Y is a covalent bond or a functional        group and E is glucuronic acid (e.g., β-glucuronic acid). Y is        preferably a functional group selected from —NH—, —CH₂—, —O—,        and —S—.

In some further embodiments, Y is a functional group as set forth above,the functional group is linked to an amino acid, and the amino acid islinked to the Stretcher unit A¹. In some embodiments, amino acid isβ-alanine. In such an embodiment, the amino acid is equivalentlyconsidered part of the Stretcher unit.

The Specificity unit L¹ and the Ligand unit are indirectly connected viathe Stretcher unit.

L¹ and A¹ may be connected by a bond selected from:

-   -   —C(═O)NH—,    -   —C(═O)O—,    -   —NHC(═O)—,    -   —OC(═O)—,    -   —OC(═O)O—,    -   —NHC(═O)O—,    -   —OC(═O)NH—, and    -   —NHC(═O)NH—.

In one embodiment, the group A¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L²        or D, the wavy line indicates the point of attachment to the        Ligand unit, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the group A¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L²        or D, the wavy line indicates the point of attachment to the        Ligand unit, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the group A¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L²        or D, the wavy line indicates the point of attachment to the        Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred        embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8,        most preferably 4 or 8.

In one embodiment, the group A¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L²        or D, the wavy line indicates the point of attachment to the        Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred        embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8,        most preferably 4 or 8.

In one embodiment, the group A¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L²        or D, the wavy line indicates the point of attachment to the        Ligand unit, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the group A¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L²        or D, the wavy line indicates the point of attachment to the        Ligand unit, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the group A¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L²        or D, the wavy line indicates the point of attachment to the        Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred        embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8,        most preferably 4 or 8.

In one embodiment, the group A¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L²        or D, the wavy line indicates the point of attachment to the        Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred        embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8,        most preferably 4 or 8.

In one embodiment, the connection between the Ligand unit and A¹ isthrough a thiol residue of the Ligand unit and a maleimide group of A¹.

In one embodiment, the connection between the Ligand unit and A¹ is:

-   -   where the asterisk indicates the point of attachment to the        remaining portion of A¹, L¹, L² or D, and the wavy line        indicates the point of attachment to the remaining portion of        the Ligand unit. In this embodiment, the S atom is typically        derived from the Ligand unit.

In each of the embodiments above, an alternative functionality may beused in place of the malemide-derived group shown below:

-   -   where the wavy line indicates the point of attachment to the        Ligand unit as before, and the asterisk indicates the bond to        the remaining portion of the A¹ group, or to L¹, L² or D.

In one embodiment, the maleimide-derived group is replaced with thegroup:

-   -   where the wavy line indicates point of attachment to the Ligand        unit, and the asterisk indicates the bond to the remaining        portion of the A¹ group, or to L¹, L² or D.

In one embodiment, the maleimide-derived group is replaced with a group,which optionally together with a Ligand unit (e.g., a Cell BindingAgent), is selected from:

-   -   —C(═O)NH—,    -   —C(═O)O—,    -   —NHC(═O)—,    -   —OC(═O)—,    -   —OC(═O)O—,    -   —NHC(═O)O—,    -   —OC(═O)NH—,    -   —NHC(═O)NH—,    -   —NHC(═O)NH,    -   —C(═O)NHC(═O)—,    -   —S—,    -   —S—S—,    -   —CH₂C(═O)—    -   —C(═O)CH₂—,    -   ═N—NH—, and    -   —NH—N═.

Of these —C(═O)CH₂— may be preferred especially when the carbonyl groupis bound to —NH—.

In one embodiment, the maleimide-derived group is replaced with a group,which optionally together with the Ligand unit, is selected from:

-   -   where the wavy line indicates either the point of attachment to        the Ligand unit or the bond to the remaining portion of the A¹        group, and the asterisk indicates the other of the point of        attachment to the Ligand unit or the bond to the remaining        portion of the A¹ group.

Other groups suitable for connecting L¹ to the Cell Binding Agent aredescribed in WO 2005/082023.

In one embodiment, the Stretcher unit A¹ is present, the Specificityunit L¹ is present and Spacer unit L² is absent. Thus, L¹ and the Drugunit are directly connected via a bond. Equivalently in this embodiment,L² is a bond.

L¹ and D may be connected by a bond selected from:

-   -   —C(═O)N<,    -   —OC(═O)N<, and    -   —NHC(═O)N<,        where N< is part of D.

In one embodiment, L¹ and D are preferably connected by a bond:

-   -   —C(═O)N<.

In one embodiment, L¹ comprises a dipeptide and one end of the dipeptideis linked to D. As described above, the amino acids in the dipeptide maybe any combination of natural amino acids and non-natural amino acids.In some embodiments, the dipeptide comprises natural amino acids. Wherethe linker is a cathepsin labile linker, the dipeptide is the site ofaction for cathepsin-mediated cleavage. The dipeptide then is arecognition site for cathepsin.

In one embodiment, the group —X₁—X₂— in dipeptide, —NH—X₁—X₂—CO—, isselected from:

-   -   -Phe-Lys-,    -   -Val-Ala-,    -   -Val-Lys-,    -   -Ala-Lys-,    -   -Val-Cit-,    -   -Phe-Cit-,    -   -Leu-Cit-,    -   -Ile-Cit-,    -   -Phe-Arg-, and    -   -Trp-Cit-;        where Cit is citrulline. In such a dipeptide, —NH— is the amino        group of X₁, and CO is the carbonyl group of X₂.

Preferably, the group —X₁—X₂— in dipeptide, —NH—X₁—X₂—CO—, is selectedfrom:

-   -   -Phe-Lys-,    -   -Val-Ala-,    -   -Val-Lys-,    -   -Ala-Lys-, and    -   -Val-Cit-.

Most preferably, the group —X₁—X₂— in dipeptide, —NH—X₁—X₂—CO—, is-Phe-Lys- or -Val-Ala-.

Other dipeptide combinations of interest include:

-   -   -Gly-Gly-,    -   -Pro-Pro-, and    -   -Val-Glu-.

Other dipeptide combinations may be used, including those describedabove.

In one embodiment, L¹-D is:

-   -   where —NH—X₁—X₂—CO is the dipeptide, —N< is part of the Drug        unit, the asterisk indicates the points of attachment to the        remainder of the Drug unit, and the wavy line indicates the        point of attachment to the remaining portion of L¹ or the point        of attachment to A¹. Preferably, the wavy line indicates the        point of attachment to A¹.

In one embodiment, the dipeptide is valine-alanine and L¹-D is:

-   -   where the asterisks, —N< and the wavy line are as defined above.

In one embodiment, the dipeptide is phenylalanine-lysine and L¹-D is:

-   -   where the asterisks, —N< and the wavy line are as defined above.

In one embodiment, the dipeptide is valine-citrulline.

In one embodiment, the groups A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the Ligand        unit, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the groups A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the Ligand        unit, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the groups A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the Ligand        unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment,        n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most        preferably 4 or 8.

In one embodiment, the groups A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the Ligand        unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment,        n is 1 and m is 0 to 10, 1 to 7, preferably 3 to 7, most        preferably 3 or 7.

In one embodiment, the groups A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the Ligand        unit, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the groups A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the Ligand        unit, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the groups A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the Ligand        unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment,        n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most        preferably 4 or 8.

In one embodiment, the groups A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the Ligand        unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment,        n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most        preferably 4 or 8.

In one embodiment, the groups L- A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        S is a sulfur group of the Ligand unit, the wavy line indicates        the point of attachment to the rest of the Ligand unit, and n is        0 to 6. In one embodiment, n is 5.

In one embodiment, the group L-A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        S is a sulfur group of the Ligand unit, the wavy line indicates        the point of attachment to the remainder of the Ligand unit, and        n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the groups L-A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        S is a sulfur group of the Ligand unit, the wavy line indicates        the point of attachment to the remainder of the Ligand unit, n        is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1        and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4        or 8.

In one embodiment, the groups L-A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the Ligand        unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment,        n is 1 and m is 0 to 10, 1 to 7, preferably 4 to 8, most        preferably 4 or 8.

In one embodiment, the groups L-A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the remainder        of the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the groups L-A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the remainder        of the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the groups L-A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the remainder        of the Ligand unit, n is 0 or 1, and m is 0 to 30. In a        preferred embodiment, n is 1 and m is 0 to 10, 1 to 8,        preferably 4 to 8, most preferably 4 or 8.

In one embodiment, the groups L-A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the remainder        of the Ligand unit, n is 0 or 1, and m is 0 to 30. In a        preferred embodiment, n is 1 and m is 0 to 10, 1 to 8,        preferably 4 to 8, most preferably 4 or 8.

In one embodiment, the Stretcher unit is an acetamide unit, having theformula:

-   -   where the asterisk indicates the point of attachment to the        remainder of the Stretcher unit, L¹ or D, and the wavy line        indicates the point of attachment to the Ligand unit.

Linker-Drugs

In other embodiments, Linker-Drug compounds are provided for conjugationto a Ligand unit. In one embodiment, the Linker-Drug compounds aredesigned for connection to a Cell Binding Agent.

In one embodiment, the Drug Linker compound has the formula:

-   -   where the asterisk indicates the point of attachment to the Drug        unit (D, as defined above), G¹ is a Stretcher group (A¹) to form        a connection to a Ligand unit, L¹ is a Specificity unit, L² (a        Spacer unit) is a covalent bond or together with —OC(═O)— forms        a self-immolative group(s).

In another embodiment, the Drug Linker compound has the formula:

G¹-L¹-L²-*

-   -   where the asterisk indicates the point of attachment to the Drug        unit (D), G¹ is a Stretcher unit (A¹) to form a connection to a        Ligand unit, L¹ is a Specificity unit, L² (a Spacer unit) is a        covalent bond or a self-immolative group(s).

L¹ and L² are as defined above. References to connection to A¹ can beconstrued here as referring to a connection to G¹.

In one embodiment, where L¹ comprises an amino acid, the side chain ofthat amino acid may be protected. Any suitable protecting group may beused. In one embodiment, the side chain protecting groups are removablewith other protecting groups in the compound, where present. In otherembodiments, the protecting groups may be orthogonal to other protectinggroups in the molecule, where present.

Suitable protecting groups for amino acid side chains include thosegroups described in the Novabiochem Catalog 2006/2007. Protecting groupsfor use in a cathepsin labile linker are also discussed in Dubowchik etal.

In certain embodiments of the invention, the group L¹ includes a Lysamino acid residue. The side chain of this amino acid may be protectedwith a Boc or Alloc protected group. A Boc protecting group is mostpreferred.

The functional group G¹ forms a connecting group upon reaction with aLigand unit (e.g., a cell binding agent.

In one embodiment, the functional group G¹ is or comprises an amino,carboxylic acid, hydroxy, thiol, or maleimide group for reaction with anappropriate group on the Ligand unit. In a preferred embodiment, G¹comprises a maleimide group.

In one embodiment, the group G¹ is an alkyl maleimide group. This groupis suitable for reaction with thiol groups, particularly cysteine thiolgroups, present in the cell binding agent, for example present in anantibody.

In one embodiment, the group G¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L²        or D, and n is 0 to 6.

In one embodiment, n is 5.

In one embodiment, the group G¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L²        or D, and n is 0 to 6.

In one embodiment, n is 5.

In one embodiment, the group G¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L²        or D n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n        is 1 and m is 0 to 10, 1 to 2, preferably 4 to 8, and most        preferably 4 or 8.

In one embodiment, the group G¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L¹,        L² or D, n is 0 or 1, and m is 0 to 30. In a preferred        embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8,        and most preferably 4 or 8.

In one embodiment, the group G¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L²        or D, and n is 0 to 6.

In one embodiment, n is 5.

In one embodiment, the group G¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L²        or D, and n is 0 to 6.

In one embodiment, n is 5.

In one embodiment, the group G¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L²        or D, n is 0 or 1, and m is 0 to 30. In a preferred embodiment,        n is 1 and m is 0 to 10, 1 to 2, preferably 4 to 8, and most        preferably 4 or 8.

In one embodiment, the group G¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L²        or D, n is 0 or 1, and m is 0 to 30. In a preferred embodiment,        n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, and most        preferably 4 or 8.

In each of the embodiments above, an alternative functionality may beused in place of the malemide group shown below:

-   -   where the asterisk indicates the bond to the remaining portion        of the G group.

In one embodiment, the maleimide-derived group is replaced with thegroup:

-   -   where the asterisk indicates the bond to the remaining portion        of the G group.

In one embodiment, the maleimide group is replaced with a group selectedfrom:

-   -   —C(═O)OH,    -   —OH,    -   —NH₂,    -   —SH,    -   —C(═O)CH₂X, where X is Cl, Br or I,    -   —CHO,    -   —NHNH₂    -   —C≡CH, and    -   —N₃ (azide).

Of these, —C(═O)CH₂X may be preferred, especially when the carbonylgroup is bound to —NH—.

In one embodiment, L¹ is present, and G¹ is —NH₂, —NHMe, —COOH, —OH or—SH.

In one embodiment, where L¹ is present, G¹ is —NH₂ or —NHMe. Eithergroup may be the N-terminal of an L¹ amino acid sequence.

In one embodiment, L¹ is present and G¹ is —NH₂, and L¹ is an amino acidsequence —X₁—X₂—, as defined above.

In one embodiment, L¹ is present and G¹ is COOH. This group may be theC-terminal of an L¹ amino acid sequence.

In one embodiment, L¹ is present and G¹ is OH.

In one embodiment, L¹ is present and G¹ is SH.

The group G¹ may be convertable from one functional group to another. Inone embodiment, L¹ is present and G¹ is —NH₂. This group is convertableto another group G¹ comprising a maleimide group. For example, the group—NH₂ may be reacted with an acids or an activated acid (e.g.,N-succinimide forms) of those G¹ groups comprising maleimide shownabove.

The group G¹ may therefore be converted to a functional group that ismore appropriate for reaction with a Ligand unit.

As noted above, in one embodiment, L¹ is present and G¹ is —NH₂, —NHMe,—COOH, —OH or —SH. In a further embodiment, these groups are provided ina chemically protected form. The chemically protected form is thereforea precursor to the linker that is provided with a functional group.

In one embodiment, G¹ is —NH₂ in a chemically protected form. The groupmay be protected with a carbamate protecting group. The carbamateprotecting group may be selected from the group consisting of:

-   -   Alloc, Fmoc, Boc, Troc, Teoc, Cbz and PNZ.

Preferably, where G¹ is —NH₂, it is protected with an Alloc or Fmocgroup.

In one embodiment, where G¹ is —NH₂, it is protected with an Fmoc group.

In one embodiment, the protecting group is the same as the carbamateprotecting group of the capping group.

In one embodiment, the protecting group is not the same as the carbamateprotecting group of the capping group. In this embodiment, it ispreferred that the protecting group is removable under conditions thatdo not remove the carbamate protecting group of the capping group.

The chemical protecting group may be removed to provide a functionalgroup to form a connection to a Ligand unit. Optionally, this functionalgroup may then be converted to another functional group as describedabove.

In one embodiment, the active group is an amine. This amine ispreferably the N-terminal amine of a peptide, and may be the N-terminalamine of the preferred dipeptides of the invention.

The active group may be reacted to yield the functional group that isintended to form a connection to a Ligand unit.

In other embodiments, the Linker unit is a precursor to the Linker unithaving an active group. In this embodiment, the Linker unit comprisesthe active group, which is protected by way of a protecting group. Theprotecting group may be removed to provide the Linker unit having anactive group.

Where the active group is an amine, the protecting group may be an amineprotecting group, such as those described in Green and Wuts.

The protecting group is preferably orthogonal to other protectinggroups, where present, in the Linker unit.

In one embodiment, the protecting group is orthogonal to the cappinggroup. Thus, the active group protecting group is removable whilstretaining the capping group. In other embodiments, the protecting groupand the capping group is removable under the same conditions as thoseused to remove the capping group.

In one embodiment, the Linker unit is:

-   -   where the asterisk indicates the point of attachment to the Drug        unit, and the wavy line indicates the point of attachment to the        remaining portion of the Linker unit, as applicable or the point        of attachment to G¹. Preferably, the wavy line indicates the        point of attachment to G¹.

In one embodiment, the Linker unit is:

where the asterisk and the wavy line are as defined above.

Other functional groups suitable for use in forming a connection betweenL¹ and the Cell Binding Agent are described in WO 2005/082023.

Ligand Unit

The Ligand Unit may be of any kind, and include a protein, polypeptide,peptide and a non-peptidic agent that specifically binds to a targetmolecule. In some embodiments, the Ligand unit may be a protein,polypeptide or peptide. In some embodiments, the Ligand unit may be acyclic polypeptide. These Ligand units can include antibodies or afragment of an antibody that contains at least one targetmolecule-binding site, lymphokines, hormones, growth factors, or anyother cell binding molecule or substance that can specifically bind to atarget. The ligand Unit is also referred to herein as a “binding agent”or “targeting agent”.

The terms “specifically binds” and “specific binding” refer to thebinding of an antibody or other protein, polypeptide or peptide to apredetermined molecule (e.g., an antigen).

Typically, the antibody or other molecule binds with an affinity of atleast about 1×10⁷ M⁻¹, and binds to the predetermined molecule with anaffinity that is at least two-fold greater than its affinity for bindingto a non-specific molecule (e.g., BSA, casein) other than thepredetermined molecule or a closely-related molecule.

Examples of Ligand units include those agents described for use in WO2007/085930, which is incorporated herein in its entirety and for allpurposes.

In some embodiments, the Ligand unit is a Cell Binding Agent that bindsto an extracellular target on a cell. Such a Cell Binding Agent can be aprotein, polypeptide, peptide or a non-peptidic agent. In someembodiments, the Cell Binding Agent may be a protein, polypeptide orpeptide. In some embodiments, the Cell Binding Agent may be a cyclicpolypeptide. The Cell Binding Agent also may be antibody or anantigen-binding fragment of an antibody. Thus, in one embodiment, thepresent invention provides an antibody-drug conjugate (ADC).

In one embodiment the antibody is a monoclonal antibody; chimericantibody; humanized antibody; fully human antibody; or a single chainantibody. One embodiment the antibody is a fragment of one of theseantibodies having biological activity. Examples of such fragmentsinclude Fab, Fab′, F(ab′)₂ and Fv fragments.

The antibody may be a diabody, a domain antibody (DAB) or a single chainantibody.

In one embodiment, the antibody is a monoclonal antibody.

Antibodies for use in the present invention include those antibodiesdescribed in WO 2005/082023 which is incorporated by reference herein inits entirety and for all purposes. Particularly preferred are thoseantibodies for tumour-associated antigens. Examples of those antigensknown in the art include, but are not limited to, thosetumour-associated antigens set out in WO 2005/082023. See, for instance,pages 41-55.

In some embodiments, the conjugates are designed to target tumour cellsvia their cell surface antigens. The antigens may be cell surfaceantigens which are either over-expressed or expressed at abnormal timesor cell types. Preferably, the target antigen is expressed only onproliferative cells (preferably tumour cells); however this is rarelyobserved in practice. As a result, target antigens are usually selectedon the basis of differential expression between proliferative andhealthy tissue.

Antibodies have been raised to target specific tumour related antigensincluding:

-   -   Cripto, CD19, CD20, CD22, CD30, CD33, Glycoprotein NMB, CanAg,        Her2 (ErbB2/Neu), CD56 (NCAM), CD70, CD79, CD138, PSCA, PSMA        (prostate specific membrane antigen), BCMA, E-selectin, EphB2,        Melanotransferin, Muc16 and TMEFF2. In any of the embodiments        provided herein, the Ligand unit can be a monoclonal antibody        that specifically binds to the Cripto antigen, CD19 antigen,        CD20 antigen, CD22 antigen, CD30 antigen, CD33 antigen,        Glycoprotein NMB, CanAg antigen, Her2 (ErbB2/Neu) antigen, CD56        (NCAM) antigen, CD70 antigen, CD79 antigen, CD138 antigen, PSCA,        PSMA (prostate specific membrane antigen), BCMA, E-selectin,        EphB2, Melanotransferin, Muc16 antigen or TMEFF2 antigen.

The Ligand unit is connected to the Linker unit. In one embodiment, theLigand unit is connected to A, where present, of the Linker unit.

In one embodiment, the connection between the Ligand unit and the Linkerunit is through a thioether bond.

In one embodiment, the connection between the Ligand unit and the Linkerunit is through a disulfide bond.

In one embodiment, the connection between the Ligand unit and the Linkerunit is through an amide bond.

In one embodiment, the connection between the Ligand unit and the Linkerunit is through an ester bond.

In one embodiment, the connection between the Ligand unit and the Linkeris formed between a thiol group of a cysteine residue of the Ligand unitand a maleimide group of the Linker unit.

The cysteine residues of the Ligand unit may be available for reactionwith the functional group of the Linker unit to form a connection. Inother embodiments, for example where the Ligand unit is an antibody, thethiol groups of the antibody may participate in interchain disulfidebonds. These interchain bonds may be converted to free thiol groups bye.g. treatment of the antibody with DTT prior to reaction with thefunctional group of the Linker unit.

In some embodiments, the cysteine residue is introduced into the heavyor light chain of an antibody. Positions for cysteine insertion bysubstitution in antibody heavy or light chains include those describedin Published U.S. Application No. 2007-0092940 and International PatentPublication WO2008/070593, which are herein incorporated by reference intheir entirety and for all purposes.

Methods of Treatment

The compounds and conjugates of the present invention may be used in amethod of therapy. Also provided is a method of treatment, comprisingadministering to a subject in need of treatment atherapeutically-effective amount of a compound or conjugate disclosedherein. The term “therapeutically effective amount” is an amountsufficient to show benefit to a patient. Such benefit may be at leastamelioration of at least one symptom. The actual amount administered,and rate and time-course of administration, will depend on the natureand severity of what is being treated. Prescription of treatment, e.g.decisions on dosage, is within the responsibility of generalpractitioners and other medical doctors.

A compound or conjugate may be administered alone or in combination withother treatments, either simultaneously or sequentially dependent uponthe condition to be treated. Examples of treatments and therapiesinclude, but are not limited to, chemotherapy (the administration ofactive agents, including, e.g. drugs; surgery; and radiation therapy.

Pharmaceutical compositions according to the present invention, and foruse in accordance with the present invention, may comprise, in additionto the active ingredient, i.e. a compound or conjugate disclosed herein,a pharmaceutically acceptable excipient, carrier, buffer, stabiliser orother materials well known to those skilled in the art. Such materialsshould be non-toxic and should not interfere with the efficacy of theactive ingredient. The precise nature of the carrier or other materialwill depend on the route of administration, which may be oral, or byinjection, e.g. cutaneous, subcutaneous, or intravenous.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may comprise a solid carrier oran adjuvant. Liquid pharmaceutical compositions generally comprise aliquid carrier such as water, petroleum, animal or vegetable oils,mineral oil or synthetic oil. Physiological saline solution, dextrose orother saccharide solution or glycols such as ethylene glycol, propyleneglycol or polyethylene glycol may be included. A capsule may comprise asolid carrier such a gelatin.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the active ingredient will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,Lactated Ringer's Injection. Preservatives, stabilisers, buffers,antioxidants and/or other additives may be included, as required.

The Compounds and Conjugates can be used to treat proliferative diseaseand autoimmune disease. The term “proliferative disease” pertains to anunwanted or uncontrolled cellular proliferation of excessive or abnormalcells which is undesired, such as, neoplastic or hyperplastic growth,whether in vitro or in vivo.

Examples of proliferative conditions include, but are not limited to,benign, pre-malignant, and malignant cellular proliferation, includingbut not limited to, neoplasms and tumours (e.g., histocytoma, glioma,astrocyoma, osteoma), cancers (e.g. lung cancer, small cell lung cancer,gastrointestinal cancer, bowel cancer, colon cancer, breast carinoma,ovarian carcinoma, prostate cancer, testicular cancer, liver cancer,kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma,osteosarcoma, Kaposi's sarcoma, melanoma), leukemias, psoriasis, bonediseases, fibroproliferative disorders (e.g. of connective tissues), andatherosclerosis. Other cancers of interest include, but are not limitedto, haematological; malignancies such as leukemias and lymphomas, suchas non-Hodgkin lymphoma, and subtypes such as DLBCL, marginal zone,mantle zone, and follicular, Hodgkin lymphoma, AML, and other cancers ofB or T cell origin.

Examples of autoimmune disease include the following: rheumatoidarthritis, autoimmune demyelinative diseases (e.g., multiple sclerosis,allergic encephalomyelitis), psoriatic arthritis, endocrineophthalmopathy, uveoretinitis, systemic lupus erythematosus, myastheniagravis, Graves' disease, glomerulonephritis, autoimmune hepatologicaldisorder, inflammatory bowel disease (e.g., Crohn's disease),anaphylaxis, allergic reaction, Sjögren's syndrome, type I diabetesmellitus, primary biliary cirrhosis, Wegener's granulomatosis,fibromyalgia, polymyositis, dermatomyositis, multiple endocrine failure,Schmidt's syndrome, autoimmune uveitis, Addison's disease, adrenalitis,thyroiditis, Hashimoto's thyroiditis, autoimmune thyroid disease,pernicious anemia, gastric atrophy, chronic hepatitis, lupoid hepatitis,atherosclerosis, subacute cutaneous lupus erythematosus,hypoparathyroidism, Dressler's syndrome, autoimmune thrombocytopenia,idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigusvulgaris, pemphigus, dermatitis herpetiformis, alopecia arcata,pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome(calcinosis, Raynaud's phenomenon, esophageal dysmotility,sclerodactyly, and telangiectasia), male and female autoimmuneinfertility, ankylosing spondolytis, ulcerative colitis, mixedconnective tissue disease, polyarteritis nedosa, systemic necrotizingvasculitis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome,Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrentabortion, anti-phospholipid syndrome, farmer's lung, erythemamultiforme, post cardiotomy syndrome, Cushing's syndrome, autoimmunechronic active hepatitis, bird-fancier's lung, toxic epidermalnecrolysis, Alport's syndrome, alveolitis, allergic alveolitis,fibrosing alveolitis, interstitial lung disease, erythema nodosum,pyoderma gangrenosum, transfusion reaction, Takayasu's arteritis,polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cellarteritis, ascariasis, aspergillosis, Sampter's syndrome, eczema,lymphomatoid granulomatosis, Behcet's disease, Caplan's syndrome,Kawasaki's disease, dengue, encephalomyelitis, endocarditis,endomyocardial fibrosis, endophthalmitis, erythema elevatum et diutinum,psoriasis, erythroblastosis fetalis, eosinophilic faciitis, Shulman'ssyndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis,heterochronic cyclitis, Fuch's cyclitis, IgA nephropathy,Henoch-Schonlein purpura, graft versus host disease, transplantationrejection, cardiomyopathy, Eaton-Lambert syndrome, relapsingpolychondritis, cryoglobulinemia, Waldenstrom's macroglobulemia, Evan'ssyndrome, and autoimmune gonadal failure.

In some embodiments, the autoimmune disease is a disorder of Blymphocytes (e.g., systemic lupus erythematosus, Goodpasture's syndrome,rheumatoid arthritis, and type I diabetes), Th1-lymphocytes (e.g.,rheumatoid arthritis, multiple sclerosis, psoriasis, Sjögren's syndrome,Hashimoto's thyroiditis, Graves' disease, primary biliary cirrhosis,Wegener's granulomatosis, tuberculosis, or graft versus host disease),or Th2-lymphocytes (e.g., atopic dermatitis, systemic lupuserythematosus, atopic asthma, rhinoconjunctivitis, allergic rhinitis,Omenn's syndrome, systemic sclerosis, or chronic graft versus hostdisease). Generally, disorders involving dendritic cells involvedisorders of Th1-lymphocytes or Th2-lymphocytes. In some embodiments,the autoimmunie disorder is a T cell-mediated immunological disorder.

In some embodiments, the amount of the Conjugate administered rangesfrom about 0.01 to about 10 mg/kg per dose. In some embodiments, theamount of the Conjugate administered ranges from about 0.01 to about 5mg/kg per dose. In some embodiments, the amount of the Conjugateadministered ranges from about 0.05 to about 5 mg/kg per dose. In someembodiments, the amount of the Conjugate administered ranges from about0.1 to about 5 mg/kg per dose. In some embodiments, the amount of theConjugate administered ranges from about 0.1 to about 4 mg/kg per dose.In some embodiments, the amount of the Conjugate administered rangesfrom about 0.05 to about 3 mg/kg per dose. In some embodiments, theamount of the Conjugate administered ranges from about 0.1 to about 3mg/kg per dose. In some embodiments, the amount of the Conjugateadministered ranges from about 0.1 to about 2 mg/kg per dose.

Includes Other Forms

Unless otherwise specified, included in the above are the well knownionic, salt, solvate, and protected forms of these substituents. Forexample, a reference to carboxylic acid (—COOH) also includes theanionic (carbon/late) form (—COO), a salt or solvate thereof, as well asconventional protected forms. Similarly, a reference to an amino groupincludes the protonated form (—N⁺HR¹R²), a salt or solvate of the aminogroup, for example, a hydrochloride salt, as well as conventionalprotected forms of an amino group. Similarly, a reference to a hydroxylgroup also includes the anionic form (—O⁻), a salt or solvate thereof,as well as conventional protected forms.

Salts

It may be convenient or desirable to prepare, purify, and/or handle acorresponding salt of the active compound, for example, apharmaceutically-acceptable salt. Examples of pharmaceuticallyacceptable salts are discussed in Berge, et al., J. Pharm. Sci., 66,1-19 (1977).

For example, if the compound is anionic, or has a functional group whichmay be anionic (e.g. —COOH may be —COO), then a salt may be formed witha suitable cation. Examples of suitable inorganic cations include, butare not limited to, alkali metal ions such as Na⁺ and K⁺, alkaline earthcations such as Ca²⁺ and Mg²⁺, and other cations such as Al⁺³. Examplesof suitable organic cations include, but are not limited to, ammoniumion (i.e. NH₄ ⁺) and substituted ammonium ions (e.g. NH₃R⁺, NH₂R₂ ⁺,NHR₃ ⁺, NR₄ ⁺). Examples of some suitable substituted ammonium ions arethose derived from: ethylamine, diethylamine, dicyclohexylamine,triethylamine, butylamine, ethylenediamine, ethanolamine,diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline,meglumine, and tromethamine, as well as amino acids, such as lysine andarginine. An example of a common quaternary ammonium ion is N(CH₃)₄ ⁺.

If the compound is cationic, or has a functional group which may becationic (e.g. —NH₂ may be —NH₃ ⁺), then a salt may be formed with asuitable anion. Examples of suitable inorganic anions include, but arenot limited to, those derived from the following inorganic acids:hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric,nitrous, phosphoric, and phosphorous.

Examples of suitable organic anions include, but are not limited to,those derived from the following organic acids: 2-acetyoxybenzoic,acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric,edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic,gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalenecarboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic,methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic,phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic,succinic, sulfanilic, tartaric, toluenesulfonic, and valeric. Examplesof suitable polymeric organic anions include, but are not limited to,those derived from the following polymeric acids: tannic acid,carboxymethyl cellulose.

Solvates

It may be convenient or desirable to prepare, purify, and/or handle acorresponding solvate of the active compound. The term “solvate” is usedherein in the conventional sense to refer to a complex of solute (e.g.active compound, salt of active compound) and solvent. If the solvent iswater, the solvate may be conveniently referred to as a hydrate, forexample, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.

Carbinolamines

The invention includes compounds where a solvent adds across the iminebond of the PBD moiety, which is illustrated below where the solvent iswater or an alcohol (R^(A)OH, where R^(A) is C₁₋₄ alkyl):

These forms can be called the carbinolamine and carbinolamine etherforms of the PBD. The balance of these equilibria depend on theconditions in which the compounds are found, as well as the nature ofthe moiety itself.

These particular compounds may be isolated in solid form, for example,by lyophilisation.

Isomers

Certain compounds may exist in one or more particular geometric,optical, enantiomeric, diasteriomeric, epimeric, atropic,stereoisomeric, tautomeric, conformational, or anomeric forms, includingbut not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, andr-forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d-and l-forms; (+) and (−) forms; keto-, enol-, and enolate-forms; syn-and anti-forms; synclinal- and anticlinal-forms; α- and β-forms; axialand equatorial forms; boat-, chair-, twist-, envelope-, andhalfchair-forms; and combinations thereof, hereinafter collectivelyreferred to as “isomers” (or “isomeric forms”).

Note that, except as discussed below for tautomeric forms, specificallyexcluded from the term “isomers”, as used herein, are structural (orconstitutional) isomers (i.e. isomers which differ in the connectionsbetween atoms rather than merely by the position of atoms in space). Forexample, a reference to a methoxy group, —OCH₃, is not to be construedas a reference to its structural isomer, a hydroxymethyl group, —CH₂OH.Similarly, a reference to ortho-chlorophenyl is not to be construed as areference to its structural isomer, meta-chlorophenyl. However, areference to a class of structures may well include structurallyisomeric forms falling within that class (e.g. C₁₋₇ alkyl includesn-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl;methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).

The above exclusion does not pertain to tautomeric forms, for example,keto-, enol-, and enolate-forms, as in, for example, the followingtautomeric pairs: keto/enol (illustrated below), imine/enamine,amide/imino alcohol, amidine/amidine, nitroso/oxime,thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.

Note that specifically included in the term “isomer” are compounds withone or more isotopic substitutions. For example, H may be in anyisotopic form, including ¹H, ²H (D), and ³H (T); C may be in anyisotopic form, including ¹²C, ¹³C, and ¹⁴C; O may be in any isotopicform, including ¹⁶O and ¹⁸O; and the like.

Unless otherwise specified, a reference to a particular compoundincludes all such isomeric forms, including (wholly or partially)racemic and other mixtures thereof. Methods for the preparation (e.g.asymmetric synthesis) and separation (e.g. fractional crystallisationand chromatographic means) of such isomeric forms are either known inthe art or are readily obtained by adapting the methods taught herein,or known methods, in a known manner.

General Synthetic Routes

The synthesis of PBD compounds is extensively discussed in the followingreferences, which discussions are incorporated herein by reference intheir entirety and for all purposes:

-   a) WO 00/12508 (pages 14 to 30);-   b) WO 2005/023814 (pages 3 to 10);-   c) WO 2004/043963 (pages 28 to 29); and-   d) WO 2005/085251 (pages 30 to 39).

Synthesis Route

The compounds of the present invention, where R¹⁰ and R¹¹ form anitrogen-carbon double bond between the nitrogen and carbon atoms towhich they are bound, can be synthesised from a compound of Formula 2:

where n is 0 or 1, and R² and R¹² represent the C2 aromatic groups ofthe compounds of the present invention, as shown in the table below:

R² R¹² n C1

0 C2

1 C3

1 C4

1 C5

1 C6

1Prot^(N) is a nitrogen protecting group for synthesis and Prot^(◯) is aprotected oxygen group for synthesis or an oxo group, by deprotectingthe imine bond by standard methods.

The compound produced may be in its carbinolamine or carbinolamine etherform depending on the solvents used. For example if Prot^(N) is Troc andProt^(◯) is an oxygen protecting group for synthesis, then thedeprotection is carried out using a Cd/Pb couple to yield the compoundof the present invention. If Prot^(N) is SEM, or an analogous group, andProt^(◯) is an oxo group, then the oxo group can be removed byreduction, which leads to a protected carbinolamine intermediate, whichcan then be treated to remove the SEM protecting group, followed by theelimination of water. The reduction of the compound of Formula 2 can beaccomplished by, for example, superhydride or lithium tetraborohydride,whilst a suitable means for removing the SEM protecting group istreatment with silica gel.

Compounds of formula 2 can be synthesised from a compound of formula 3a:

where R², Prot^(N) and Prot^(◯) are as defined for compounds of formula2, by coupling an organometallic derivative comprising R¹², such as anorganoboron derivative. The organoboron derivative may be a boronate orboronic acid.

Compounds of formula 2 can be synthesised from a compound of formula 3b:

where R¹², Prot^(N) and Prot^(◯) are as defined for compounds of formula2, by coupling an organometallic derivative comprising R², such as anorganoboron derivative. The organoboron derivative may be a boronate orboronic acid.

Compounds of formulae 3a and 3b can be synthesised from a compound offormula 4:

where Prot^(N) and Prot^(◯) are as defined for compounds of formula 2,by coupling about a single equivalent (e.g. 0.9 or 1 to 1.1 or 1.2) ofan organometallic derivative, such as an organoboron derivative,comprising R² or R¹².

The couplings described above are usually carried out in the presence ofa palladium catalyst, for example Pd(PPh₃)₄, Pd(OCOCH₃)₂, PdCl₂,Pd₂(dba)₃. The coupling may be carried out under standard conditions, ormay also be carried out under microwave conditions.

The two coupling steps are usually carried out sequentially. They may becarried out with or without purification between the two steps. If nopurification is carried out, then the two steps may be carried out inthe same reaction vessel. Purification is usually required after thesecond coupling step. Purification of the compound from the undesiredby-products may be carried out by column chromatography or ion-exchangeseparation.

The synthesis of compounds of formula 4 where Prot^(◯) is an oxo groupand Prot^(N) is SEM are described in detail in WO 00/12508, which isincorporated herein by reference in its entirety and for all purposes.In particular, reference is made to scheme 7 on page 24, where the abovecompound is designated as intermediate P. This method of synthesis isalso described in WO 2004/043963. Further reference is also made to thesynthesis of compounds 8a and 8b in WO 2010/043880 (pages 36 to 45),which is incorporated herein by reference in its entirety and for allpurposes.

The synthesis of compounds of formula 4 where Prot^(◯) is a protectedoxygen group for synthesis are described in WO 2005/085251, whichsynthesis is herein incorporated by reference in its entirety and forall purposes.

Compounds of the present invention where R¹⁰ and R^(10′) are H and R¹¹and R^(11′) are SO_(z)M, can be synthesised from compounds of thepresent invention where R¹⁰ and R¹¹ form a nitrogen-carbon double bondbetween the nitrogen and carbon atoms to which they are bound, by theaddition of the appropriate bisulphite salt or sulphinate salt, followedby an appropriate purification step. Further methods are described in GB2 053 894, which is herein incorporated by reference.

In some embodiments of the invention, it may be that the compounds ofFormula 2 are used in the synthesis of the drug linker compounds. Inthese embodiments, the removal of the N10/C11 protecting groups mayoccur during the synthesis of the drug linker compounds.

Nitrogen Protecting Groups for Synthesis

Nitrogen protecting groups for synthesis are well known in the art. Inthe present invention, the protecting groups of particular interest arecarbamate nitrogen protecting groups and hemi-aminal nitrogen protectinggroups.

Carbamate nitrogen protecting groups have the following structure:

wherein R′¹⁰ is R as defined above. A large number of suitable groupsare described on pages 503 to 549 of Greene, T. W. and Wuts, G. M.,Protective Groups in Organic Synthesis, 3^(rd) Edition, John Wiley &Sons, Inc., 1999, which is incorporated herein by reference in itsentirety and for all purposes.

Particularly preferred protecting groups include Troc, Teoc, Fmoc, BOC,Doc, Hoc, TcBOC, 1-Adoc and 2-Adoc.

Other possible groups are nitrobenzyloxycarbonyl (e.g.4-nitrobenzyloxycarbonyl) and 2-(phenylsulphonyl)ethoxycarbonyl.

Those protecting groups which can be removed with palladium catalysisare not preferred, e.g. Alloc.

Hemi-aminal nitrogen protecting groups have the following structure:

wherein R′¹⁰ is R as defined above. A large number of suitable groupsare described on pages 633 to 647 as amide protecting groups of Greene,T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3^(rd)Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein byreference in its entirety and for all purposes. The groups disclosedherein can be applied to compounds of the present invention. Such groupsinclude, but are not limited to, SEM, MOM, MTM, MEM, BOM, nitro ormethoxy substituted BOM, Cl₃CCH₂OCH₂—.

Protected Oxygen Group for Synthesis

Protected oxygen group for synthesis are well known in the art. A largenumber of suitable oxygen protecting groups are described on pages 23 to200 of Greene, T. W. and Wuts, G. M., Protective Groups in OrganicSynthesis, 3^(rd) Edition, John Wiley & Sons, Inc., 1999, which isincorporated herein by reference in its entirety and for all purposes.

Classes of particular interest include silyl ethers, methyl ethers,alkyl ethers, benzyl ethers, esters, acetates, benzoates, carbonates,and sulfonates.

Preferred oxygen protecting groups include acetates, TBS and THP.

Synthesis of Drug Conjugates

Conjugates comprising PBD dimers as described herein can be preparedusing the knowledge of the skilled artisan in combination with theteachings provided herein. For example, linkers are described in U.S.Pat. No. 6,214,345, U.S. Pat. No. 7,498,298 as well as WO 2009/0117531,each of which is incorporated herein by reference in their entirety andfor all purposes. Other linkers can be prepared according to thereferences cited herein or as known to the skilled artisan.

Linker-Drug compounds can be prepared according to methods known in theart in combination with the teachings provided herein. For example,linkage of amine-based X substituents (of the PBD dimer Drug unit) toactive groups of the Linker units can be performed according to methodsgenerally described in U.S. Pat. Nos. 6,214,345 and 7,498,298; and WO2009-0117531, or as otherwise known to the skilled artisan.

Antibodies can be conjugated to Linker-Drug compounds as described inDoronina et al., Nature Biotechnology, 2003, 21, 778-784). Briefly,antibodies (4-5 mg/mL) in PBS containing 50 mM sodium borate at pH 7.4are reduced with tris(carboxyethyl)phosphine hydrochloride (TCEP) at 37°C. The progress of the reaction, which reduces interchain disulfides, ismonitored by reaction with 5,5′-dithiobis(2-nitrobenzoic acid) andallowed to proceed until the desired level of thiols/mAb is achieved.The reduced antibody is then cooled to 0° C. and alkylated with 1.5equivalents of maleimide drug-linker per antibody thiol. After 1 hour,the reaction is quenched by the addition of 5 equivalents of N-acetylcysteine. Quenched drug-linker is removed by gel filtration over a PD-10column. The ADC is then sterile-filtered through a 0.22 μm syringefilter. Protein concentration can be determined by spectral analysis at280 nm and 329 nm, respectively, with correction for the contribution ofdrug absorbance at 280 nm. Size exclusion chromatography can be used todetermine the extent of antibody aggregation, and RP-HPLC can be used todetermine the levels of remaining NAC-quenched drug-linker.

Antibodies with introduced cysteine residues can be conjugated toLinker-Drug compounds as described in International Patent PublicationWO2008/070593, which is incorporated herein by reference in its entiretyand for all purposes or as follows. Antibodies containing an introducedcysteine residue in the heavy chain are fully reduced by adding 10equivalents of TCEP and 1 mM EDTA and adjusting the pH to 7.4 with 1MTris buffer (pH 9.0). Following a 1 hour incubation at 37° C., thereaction is cooled to 22° C. and 30 equivalents of dehydroascorbic acidis added to selectively reoxidize the native disulfides, while leavingthe introduced cysteine in the reduced state. The pH is adjusted to 6.5with 1M Tris buffer (pH 3.7) and the reaction is allowed to proceed for1 hour at 22° C. The pH of the solution is then raised again to 7.4 byaddition of 1 M Tris buffer (pH 9.0). 3.5 equivalents of the PBD druglinker in DMSO is placed in a suitable container for dilution withpropylene glycol prior to addition to the reaction. To maintainsolubility of the PBD drug linker, the antibody itself is first dilutedwith propylene glycol to a final concentration of 33% (e.g., if theantibody solution was in a 60 mL reaction volume, 30 mL of propyleneglycol was added). This same volume of propylene glycol (30 mL in thisexample) is added to the PBD drug linker as a diluent. After mixing, thesolution of PBD drug linker in propylene glycol is added to the antibodysolution to effect the conjugation; the final concentration of propyleneglycol is 50%. The reaction is allowed to proceed for 30 minutes andthen quenched by addition of 5 equivalents of N-acetyl cysteine. The ADCis purified by ultrafiltration through a 30 kD membrane. (Note that theconcentration of propylene glycol used in the reaction can be reducedfor any particular PBD, as its sole purpose is to maintain solubility ofthe drug linker in the aqueous media.)

For halo-acetamide-based Linker-Drug compounds, conjugation can beperformed generally as follows. To a solution of reduced and reoxidizedantibodies (having introduced cysteines in the heavy chain) in 10 mMTris (pH 7.4), 50 mM NaCl, and 2 mM DTPA is added 0.5 volumes ofpropylene glycol. A 10 mM solution of acetamide-based Linker-Drugcompound in dimethylacetamide is prepared immediately prior toconjugation. An equivalent amount of propylene glycol as added to theantibody solution is added to a 6-fold molar excess of the Linker-Drugcompound. The dilute Linker-Drug solution is added to the antibodysolution and the pH is adjusted to 8-8.5 using 1 M Tris (pH 9). Theconjugation reaction is allowed to proceed for 45 minutes at 37° C. Theconjugation is verified by reducing and denaturing reversed phase PLRP-Schromatography. Excess Linker-Drug compound is removed with Quadrasil MPresin and the buffer is exchanged into 10 mM Tris (pH 7.4), 50 mM NaCl,and 5% propylene glycol using a PD-10 desalting column.

Illustrative Synthesis Schemes for Drug Linkers

The following scheme is illustrative of routes for synthesising druglinkers.

R^(2′) represents the part of R² (as defined above) which links the PBDcore to the NH₂ group (for compound C1, the NH₂ group is replaced byNHMe). n is as defined above.

The glucuronide linker intermediate S1 (reference: Jeffrey et al.,Bioconjugate Chemistry, 2006, 17, 831-840) can be treated withdiphosgene in dichlroromethane at −78° C. to afford the glucuronidechloroformate, which is then reacted with the PBD dimer S2 dissolved inCH₂Cl₂ by dropwise addition. Warming the reaction to 0° C. over 2 hoursfollowed by extraction will yield the compound S3. Treating a solutionof S3 in an equal solvent mixture of MeOH, tetrahydrofuran, and water(cooled to 0° C.) with lithium hydroxide monohydrate for 4 hours,followed by reaction with glacial acetic acid will yield the compoundS4. Adding maleimidocaproyl NHS ester to a solution of S4 in DMF,followed by diisopropylethylamine and stirring at room temperature undernitrogen for 2 hours will yield the desired drug linker S5.

The methods of Examples 2, 3, 4, 6, 7 and 8 could be adapted for all thePBD compounds of the present invention.

Further Preferences

The following preferences may apply to all aspects of the invention asdescribed above, or may relate to a single aspect. The preferences maybe combined together in any combination.

When R¹⁰ is carbamate nitrogen protecting group, it may preferably beTeoc, Fmoc and Troc, and may more preferably be Troc.

When R¹¹ is O-Prot^(◯), wherein Prot^(◯) is an oxygen protecting group,Prot^(◯) may preferably be TBS or THP, and may more preferably be TBS.

When R¹⁰ is a hemi-aminal nitrogen protecting group, it may preferablybe MOM, BOM or SEM, and may more preferably be SEM.

Conjugates

-   (a) Conjugates of the present invention include, for example, those    of the formula:

CBA-A¹-L¹-*

-   -   where the asterisk indicates the point of attachment to the PBD        dimer (D), or the Spacer Unit, CBA is the Cell Binding Agent, L¹        is a Specificity unit that is cleavable by the action of an        enzyme, and A¹ is a Stretcher unit connecting L¹ to the Cell        Binding Agent.

-   (b) Conjugates of the present invention include, for example, those    of the formula:

CBA-A¹-L¹-*

-   -   where the asterisk indicates the point of attachment to the PBD        dimer (D), CBA is the Cell Binding Agent, A¹ is a Stretcher unit        connecting L¹ to the Cell Binding Agent and L¹ is a Specificity        unit that is cleavable by the action of cathepsin, L¹ is a        dipeptide, L¹ is a dipeptide that is cleavable by the action of        cathepsin or L¹ is a dipeptide selected from -Phe-Lys-,        -Val-Ala-, -Val-Lys-, -Ala-Lys-, and -Val-Cit-.

Preferred conjugates of the present invention include any of thosedescribed in (a) and (b) wherein A¹ is

-   -   where the asterisk indicates the point of attachment to L¹, the        wavy line indicates the point of attachment to CBA, and n is 0        to 6 (preferably n is 5).

Preferred conjugates of the present invention include those wherein theLinker Unit is

wherein the wavy line indicates the point of attachment to the Ligandunit (e.g., antibody) and the asterisk indicates the point of attachmentto D.

In a particularly preferred embodiment, for all of the conjugates, theconnection between the antibody and the Linker unit is formed between athiol group of a cysteine residue of the antibody and a maleimide groupof the Linker unit.

In a particularly preferred embodiment, for all of the preferredconjugates, the antibody is a monoclonal antibody that specificallybinds to the Cripto antigen, CD19 antigen, CD20 antigen, CD22 antigen,CD30 antigen, CD33 antigen, Glycoprotein NMB, CanAg antigen, Her2(ErbB2/Neu) antigen, CD56 (NCAM) antigen, CD70 antigen, CD79 antigen,CD138 antigen, PSCA, PSMA (prostate specific membrane antigen), BCMA,E-selectin, EphB2, Melanotransferin, Muc16 antigen or TMEFF2 antigen.

EXAMPLES General Experimental Methods for Example 1

Optical rotations were measured on an ADP 220 polarimeter (BellinghamStanley Ltd.) and concentrations (c) are given in g/100 mL. Meltingpoints were measured using a digital melting point apparatus(Electrothermal). IR spectra were recorded on a Perkin-Elmer Spectrum1000 FT IR Spectrometer. ¹H and ¹³C NMR spectra were acquired at 300 Kusing a Bruker Avance NMR spectrometer at 400 and 100 MHz, respectively.Chemical shifts are reported relative to TMS (6=0.0 ppm), and signalsare designated as s (singlet), d (doublet), t (triplet), dt (doubletriplet), dd (doublet of doublets), ddd (double doublet of doublets) orm (multiplet), with coupling constants given in Hertz (Hz). Massspectroscopy (MS) data were collected using a Waters Micromass ZQinstrument coupled to a Waters 2695 HPLC with a Waters 2996 PDA. WatersMicromass ZQ parameters used were: Capillary (kV), 3.38; Cone (V), 35;Extractor (V), 3.0; Source temperature (° C.), 100; DesolvationTemperature (° C.), 200; Cone flow rate (L/h), 50; De-solvation flowrate (L/h), 250. High-resolution mass spectroscopy (HRMS) data wererecorded on a Waters Micromass QTOF Global in positive W-mode usingmetal-coated borosilicate glass tips to introduce the samples into theinstrument. Thin Layer Chromatography (TLC) was performed on silica gelaluminium plates (Merck 60, F₂₅₄), and flash chromatography utilisedsilica gel (Merck 60, 230-400 mesh ASTM). Except for the HOBt(NovaBiochem) and solid-supported reagents (Argonaut), all otherchemicals and solvents were purchased from Sigma-Aldrich and were usedas supplied without further purification. Anhydrous solvents wereprepared by distillation under a dry nitrogen atmosphere in the presenceof an appropriate drying agent, and were stored over 4 Å molecularsieves or sodium wire. Petroleum ether refers to the fraction boiling at40−60° C.

General LC/MS conditions: The HPLC (Waters Alliance 2695) was run usinga mobile phase of water (A) (formic acid 0.1%) and acetonitrile (B)(formic acid 0.1%). Gradient: initial composition 5% B over 1.0 min then5% B to 95% B within 3 min. The composition was held for 0.5 min at 95%B, and then returned to 5% B in 0.3 minutes. Total gradient run timeequals 5 min. Flow rate 3.0 mL/min, 400 μL was split via a zero deadvolume tee piece which passes into the mass spectrometer. Wavelengthdetection range: 220 to 400 nm. Function type: diode array (535 scans).Column: Phenomenex® Onyx Monolithic C18 50×4.60 mm

Example 1

(a)(S)-2-(4-methoxyphenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(trifluoromethylsulfonyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentoxyoxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione(2)

Tetrakis-tris-phenylphosphine palladium complex (38 mg, 3.23×10⁻⁵ mol,0.02 eq) was added to a stirred, degassed mixture of1,1′-[[(Pentane-1,5-diyl)dioxy]bis(11aS)-7-methoxy-2-[[(trifluoromethyl)sulfonyl]oxy]-10-((2-(trimethylsilyl)ethoxy)methyl)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione](1)(Compound 8b in WO 2010/043880) (185 mg, 1.62 mmol, 1.0 eq.),4-methoxyphenyl boronic acid (234 mg, 1.54 mmol, 0.95 eq.) and Na₂CO₃(274 mg, 2.59 mmol, 1.6 eq.) in toluene/ethanol/water (10 mL/5 mL/5 mL).The reaction mixture was allowed to stir at room temperature under anargon atmosphere for 3 hours. The reaction mixture was diluted withethylacetate and the aqueous portion was separated. The organic portionwas washed with water, saturated brine, dried (MgSO₄) and evaporatedunder reduced pressure. Purification by flash column chromatography[gradient elution, ethylacetate 30%/n-hexane 70% to ethylacetate80%/n-hexane 20%] afforded the product as a yellow foam (0.7 g, 39%).Analytical Data: RT 3.97 min; MS (ES⁺) m/z (relative intensity) 1103([M+H]⁺, 100),

(b)(S)-2-(4-(aminomethyl)phenyl)-7-methoxy-8-((5-(((S)-7-methoxy-2-(4-methoxyphenyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H,11aH)-dione (3)

Tetrakis-tris-phenylphosphine palladium complex (30 mg, 2.5×10⁻⁵ mol,0.04 eq) was added to a stirred, degassed mixture of the methoxytriflate (2) (700 mg, 0.63 mmol, 1.0 eq.), 4-aminomethylphenyl boronicacid (190 mg, 1.015 mmol, 1.6 eq.) and Na₂CO₃ (303 mg, 2.85 mmol, 4.5eq.) in toluene/ethanol/water (20 mL/10 mL/10 mL). The reaction mixturewas heated at 75° C. under an argon atmosphere for 3 hours. The reactionmixture was diluted with ethylacetate and the aqueous portion wasseparated. The organic portion was washed with water, saturated brine,dried (MgSO₄) and evaporated under reduced pressure gave the crudeproduct as a brown foam. Purification by flash column chromatography[gradient elution, ethylacetate 50%/n-hexane 50% to ethylacetate 100%]afforded the product (0.55 g, 82%). Analytical Data: RT 3.48 min; MS(ES⁺) m/z (relative intensity) 1060 ([M+H]⁺, 100).

General Experimental Methods for Example 2

All commercially available anhydrous solvents were used without furtherpurification. Analytical thin layer chromatography was performed onsilica gel 60 F254 aluminum sheets (EMD Chemicals, Gibbstown, N.J.).Radial chromatography was performed on Chromatotron apparatus (HarrisResearch, Palo Alto, Calif.). Analytical HPLC was performed on a VarianProStar 210 solvent delivery system configured with a Varian ProStar 330PDA detector. Samples were eluted over a C12 Phenomenex Synergi 2.0×150mm, 4 μm, 80 Å reverse-phase column. The acidic mobile phase consistedof acetonitrile and water both containing either 0.05% trifluoroaceticacid or 0.1% formic acid (denoted for each compound). Compounds wereeluted with a linear gradient of acidic acetonitrile from 5% at 1 minpost injection, to 95% at 11 min, followed by isocratic 95% acetonitrileto 15 min (flow rate=1.0 mL/min). LC-MS was performed on a ZMD Micromassmass spectrometer interfaced to an HP Agilent 1100 HPLC instrumentequipped with a C12 Phenomenex Synergi 2.0×150 mm, 4 μm, 80 Å reversephase column. The acidic eluent consisted of a linear gradient ofacetonitrile from 5% to 95% in 0.1% aqueous formic acid over 10 min,followed by isocratic 95% acetonitrile for 5 min (flow rate=0.4 mL/min).Preparative HPLC was carried out on a Varian ProStar 210 solventdelivery system configured with a Varian ProStar 330 PDA detector.Products were purified over a C12 Phenomenex Synergi 10.0×250 mm, 4 μm,80 Å reverse phase column eluting with 0.1% formic acid in water(solvent A) and 0.1% formic acid in acetonitrile (solvent B). Thepurification method consisted of the following gradient of solvent A tosolvent B: 90:10 from 0 to 5 min; 90:10 to 10:90 from 5 min to 80 min;followed by isocratic 10:90 for 5 min. The flow rate was 4.6 mL/min withmonitoring at 254 nm. NMR spectral data were collected on a VarianMercury 400 MHz spectrometer. Coupling constants (J) are reported inhertz.

Example 2

(a) Allyl((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-2-(4-methoxyphenyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)benzyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(4)

A 10 mL flask was charged with alloc-Val-Ala (15 mg, 57 μmol), EEDQ (17mg, 69 μmol), and 0.72 mL anhydrous CH₂Cl₂. Methanol (40 μL) was addedto facilitate dissolution and the mixture was stirred under nitrogen for15 minutes. SEM-dilactam benzylamine 3 (40 mg, 38 μmol) was then addedand the reaction was stirred at room temperature for 4 hours, at whichtime LC-MS revealed conversion to product. The reaction wasconcentrated, dissolved in minimal CH₂Cl₂, and purified by radialchromatography on a 1 mm chromatotron plate eluted with CH₂Cl₂/MeOHmixtures (100:0 to 90:10 CH₂Cl₂/MeOH) to provide 4 (42 mg, 85%). LC-MS:t_(R) 15.50 min, m/z (ES⁺) found 1315.1 (M+H)⁺.

(b) Allyl((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)benzyl)amino)-1-oxopropan-2-ylamino)-3-methyl-1-oxobutan-2-yl)carbamate(5)

A 10 mL flame-dried flask was charged with 4 (40 mg, 30 μmol) andanhydrous THF (0.6 mL), and cooled to −78° C. Lithiumtriethylborohydride (60 μL of a 1 M THF solution) was added dropwise andthe reaction was stirred under nitrogen at −78 C for 2 h, at which timeLC-MS revealed roughly 50% conversion. An additional 30 μl of reductantwas then added and stirring continued for two additional hours, at whichtime the reaction was complete. The reaction was then quenched viaaddition of 1 mL water and warmed to room temperature, then diluted with25 mL brine and extracted three times with 25 mL dichloromethane. Thecombined organics were washed with brine, dried over sodium sulfate, andconcentrated to dryness. The residue obtained was dissolved inchloroform (0.75 mL), ethanol (2 mL, and water (0.3 mL), and 800 mg ofsilica gel was added, providing a thick slurry that was sealed andstirred at room temperature for four days. TLC analysis at that timerevealed conversion to imine 5. The silica gel was then filtered andwashed multiple times with 10% methanol in chloroform until no furtherPBD absorbance was observed in the filtrate. The combined organics werethen washed with brine, dried over sodium sulfate, and concentrated todryness. The residue was then dissolved in minimal CH₂Cl₂, and purifiedby radial chromatography on a 1 mm chromatotron plate eluted withCH₂Cl₂/MeOH mixtures (100:0 to 90:10 CH₂Cl₂/MeOH) to provide 3 (19 mg,61%). Analytical HPLC: t_(R) 11.99 min. LC-MS: t_(R) 12.76 min, m/z(ES⁺) found 1022.4 (M+H)⁺.

(c)6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N—((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)benzyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide(6)

Alloc-protected di-imine PBD 5 (19 mg, 19 μmol) was added to aflame-dried flask and dissolved in anhydrous dichloromethane (1.9 mL).Triphenylphosphine (0.25 mg, 1 μmol), pyrollidine (3.1 μL, 38 μL), andtetrakis(triphenylphosphine)palladium(0) (0.5 mg, 0.5 μmol) were added,the reaction was then stirred under nitrogen for 30 minutes at roomtemperature, at which time LC-MS revealed complete alloc-deprotection.The reaction was loaded directly onto a 1 mm chromatotron plate elutedwith CH₂Cl₂/MeOH mixtures (100:0 to 80:20 CH₂Cl₂/MeOH) to provide thefree amine (14 mg, 79%). Analytical HPLC: t_(R) 9.32 min. LC-MS: t_(R)11.61 min, m/z (ES⁺) found 938.5 (M+H)⁺. The free amine was thendissolved in anhydrous DMF (0.37 mL) and maleimidocaproyl NHS ester wasadded (6.9 mg, 22 μmol), followed by diisopropylethylamine (13 μL, 75μL). The reaction was stirred at room temperature for three hours, atwhich time LC-MS revealed complete consumption of starting material. Thereaction mixture was diluted with dichloromethane and purified by radialchromatography on a 1 mm chromatotron plate eluted with CH₂Cl₂/MeOHmixtures (100:0 to 90:10 CH₂Cl₂/MeOH) to provide 4 (15 mg, 89%).Analytical HPLC: t_(R) 11.63 min. LC-MS: t_(R) 12.73 min, m/z (ES⁺)found 1132.1 (M+H)⁺.

General Experimental Methods for Examples 3-7.

All commercially available anhydrous solvents were used without furtherpurification. Analytical thin layer chromatography was performed onsilica gel 60 F254 aluminum sheets (EMD Chemicals, Gibbstown, N.J.).Radial chromatography was performed on Chromatotron apparatus (HarrisResearch, Palo Alto, Calif.). Analytical HPLC was performed on a VarianProStar 210 solvent delivery system configured with a Varian ProStar 330PDA detector. Samples were eluted over a C12 Phenomenex Synergi 2.0×150mm, 4 μm, 80 Å reverse-phase column. The acidic mobile phase consistedof acetonitrile and water both containing either 0.05% trifluoroaceticacid or 0.1% formic acid (denoted for each compound). Compounds wereeluted with a linear gradient of acidic acetonitrile from 5% at 1 minpost injection, to 95% at 11 min, followed by isocratic 95% acetonitrileto 15 min (flow rate=1.0 mL/min). LC-MS was performed on a ZMD Micromassmass spectrometer interfaced to an HP Agilent 1100 HPLC instrumentequipped with a C12 Phenomenex Synergi 2.0×150 mm, 4 μm, 80 Å reversephase column. The acidic eluent consisted of a linear gradient ofacetonitrile from 5% to 95% in 0.1% aqueous formic acid over 10 min,followed by isocratic 95% acetonitrile for 5 min (flow rate=0.4 mL/min).Preparative HPLC was carried out on a Varian ProStar 210 solventdelivery system configured with a Varian ProStar 330 PDA detector.Products were purified over a C12 Phenomenex Synergi 10.0×250 mm, 4 μm,80 Å reverse phase column eluting with 0.1% formic acid in water(solvent A) and 0.1% formic acid in acetonitrile (solvent B). Thepurification method consisted of the following gradient of solvent A tosolvent B: 90:10 from 0 to 5 min; 90:10 to 10:90 from 5 min to 80 min;followed by isocratic 10:90 for 5 min. The flow rate was 4.6 mL/min withmonitoring at 254 nm. NMR spectral data were collected on a VarianMercury 400 MHz spectrometer. Coupling constants (J) are reported inhertz.

Example 3

(a)(S)-2-(4-(aminomethyl)phenyl-7-methoxy-8-((5-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one(7)

A flame-dried flask was charged with SEM dilactam 3 (100 mg, 94 μmol, 1eq) dissolved in anhydrous tetrahydrofuran (THF, 1.9 mL), and cooled to−78° C. Lithium triethylborohydride (0.19 mL of a 1 M solution in THF,188 μmol, 2 eq) was added dropwise and the reaction was stirred undernitrogen for 1 hour, at which time LC revealed incomplete conversion toproduct. An additional 0.1 mL of reductant was added and the reactionwas stirred for one more hour. The reaction was quenched through theaddition of water (3 mL) and allowed to warm to room temperature, thendiluted with brine (25 mL) and extracted three times withdichloromethane (25 mL). The combined organics were washed with brine(25 mL), dried over sodium sulfate, and evaporated to dryness. Theresidue was dissolved in a mixture of dichloromethane (2.4 mL), ethanol(6.2 mL), and water (0.9 mL), and silica gel (2.4 g) was added. Theresulting slurry was stirred at room temperature for 3 days. TLCanalysis revealed conversion to imine 7, at which time the slurry wasfiltered over a sintered glass funnel and the silica gel cake was washedwith 10% methanol in chloroform until no further PBD absorbance wasobserved in the filtrate. Concentration of the filtrate provided 70 mgof crude imine dimer 7, which was split and 40 mg taken forward forpurification. The material was dissolved in minimal dichloromethane andpurified by radial chromatography on a 1 mm chromatotron plate elutedwith CH₂Cl₂/MeOH mixtures (100:0 to 80:20) to provide 7 (11 mg, 27% fromsplit material). TLC: R_(f)=0.21, 20% MeOH in CH₂Cl₂. LC-MS: t_(R) 11.30min, m/z (ES⁺) found 768.3 (M+H)⁺.

(b) 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)benzylcarbamate(9)

A flame-dried flask was charged with benzylamine 7 (7.3 mg, 9.5 μmol, 1eq) dissolved in anhydrous dimethylformamide (0.2 mL).Maleimidocaproyl-Valine-Citrulline-PAB-OCO-pNP (Dubowchik et al.,Bioconjugate Chemistry, 2002, 13, 855-869) (7 mg, 9.5 μmol, 1 eq) wasadded, followed by diisopropylethylamine (16.5 μL, 95 μmol, 10 eq), thereaction was then stirred at room temperature under a nitrogenatmosphere. LC revealed conversion to product after 1.5 hours; thereaction was diluted with dichloromethane and loaded directly on to a 1mm chromatotron plate eluted with CH₂Cl₂/MeOH mixtures (100:0 to 80:20)to provide purified drug linker 9 (9.3 mg, 72%). TLC: R_(f)=0.24, 10%MeOH in CH₂Cl₂. LC-MS: t_(R) 12.61 min, m/z (ES⁺) found 1366.8 (M+H)⁺.

Example 4

(a)(2S,3R,4S,5R,6R)-2-(2-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-((((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)benzyl)carbamoyl)oxy)methyl)phenoxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyl triacetate (12)

A flame-dried flask was charged with SEM dilactam 3 (40 mg, 38 μmol, 1eq) dissolved in anhydrous tetrahydrofuran (0.8 mL), and cooled to −78°C. Lithium triethylborohydride (80 μL of a 1 M solution in THF, 76 μmol,2 eq) was added dropwise and the reaction was stirred under nitrogen for1.5 hours, at which time LC revealed incomplete conversion to product.An additional 40 μL of reductant was added and the reaction was stirredfor one more hour. The reaction was quenched through the addition ofwater (1 mL) and allowed to warm to room temperature, then diluted withbrine (25 mL) and extracted three times with dichloromethane (25 mL).The combined organics were washed with brine (25 mL), dried over sodiumsulfate, and evaporated to dryness. The SEM carbinolamine 10 (39 mg) wascarried forward without further purification. Activated glucuronidelinker 11 (Jeffrey et al., Bioconjugate Chemistry, 2006, 17, 831-840)(38 mg, 42 μmol, 1.1 eq) was dissolved in anhydrous dimethylformamide(0.6 mL) and added to a flask containing 10 (39 mg, 37 μmol, 1 eq).Diisopropylethylamine (13 μL, 74 μmol, 2 eq) was added and the reactionwas stirred at room temperature under nitrogen; after 1.5 hours LC-MSrevealed conversion to the coupled product. The reaction was dilutedwith brine (25 mL) and extracted three times with dichloromethane (25mL). The combined organics were washed with brine (25 mL), dried oversodium sulfate, and evaporated to dryness. The residue obtained wasdissolved in a mixture of chloroform (0.7 mL), ethanol (1.25 mL), andwater (0.17 mL), and silica gel (1 g) was added. The resulting slurrywas stirred at room temperature for 3 days. TLC analysis revealedconversion to drug-linker 12, at which time the slurry was filtered overa sintered glass funnel and the silica gel cake was washed with 10%methanol in chloroform until no further PBD absorbance was observed inthe filtrate. Concentration of the filtrate provided crude product 12.The material was dissolved in minimal dichloromethane and purified byradial chromatography on a 1 mm chromatotron plate eluted withCH₂Cl₂/MeOH mixtures (100:0 to 80:20) to provide 12 (25 mg, 36%). LC-MS:m/z (ES⁺) found 1542.9 (M+H)⁺.

(b)(2S,3S,4S,5R,6S)-6-(2-(3-aminopropanamido)-4-((((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)benzylcarbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicacid (13)

Protected glucuronide linker 12 (25 mg, 16 μmol, 1 eq) was dissolved inmethanol (0.5 mL) and tetrahydrofuran (0.5 mL), and the cooled to 0° C.Lithium hydroxide monohydrate (4 mg, 96 μmol, 6 eq) was dissolved inwater (0.5 mL) and added dropwise to the reaction, which was thenallowed to warm to room temperature and monitored by LC-MS. AdditionalLiOH (3.2 mg, 76 μmol, 4.8 eq) in 0.4 mL of water was added to thereaction after 2 h to further drive conversion to product. Glacialacetic acid (11 μL, 195 μmol, 12 eq) was added, followed by 1 mL ofdimethylsulfoxide, the volatile solvents were then removed by rotaryevaporation. The crude product was purified by preparative HPLC toprovide deprotected glucuronide linker 13 (2 mg, 11%). LC-MS: t_(R)11.54 min, m/z (ES⁺) found 1180.0 (M+H)+.

(c)(2S,3S,4S,5R,6S)-6-(2-(3-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)propanamido)-4-((((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)benzyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicacid (14)

A flame-dried flask was charged with glucuronide linker 13 (2 mg, 1.7μmol, 1 eq), maleimidocaproic acid N-hydroxysuccinimide ester (0.8 mg,2.6 μmol, 1.5 eq), and anhydrous dimethylformamide (85 μL).Diisopropylethylamine (1.5 uL, 8.5 μmol, 5 eq) was added, the reactionwas then stirred at room temperature under nitrogen. After 2 hours HPLCrevealed conversion to product. The reaction was diluted indimethylsulfoxide and purified by preparative HPLC to provide PBDglucuronide linker 14 (1.4 mg, 61%). LC-MS: t_(R) 12.30 min, m/z (ES⁺)found 1373.7 (M+H)⁺.

Example 5

(a)(S)-8-((5-(((S)-2-(4-aminophenyl)-7-methoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yltrifluoromethanesulfonate (15)

A flask was charged with bis triflate 1 (500 mg, 437 μmol, 1 eq)dissolved in toluene (6.5 mL), ethanol (3.2 mL), and water (3.2 mL). Tothe stirred solution was added 4-aminophenylboronic acid pinacol ester(87 mg, 398 μmol, 0.91 eq), sodium carbonate (213 mg, 2.0 mmol, 4.6 eq),and tetrakis(triphenylphosphine)palladium(0) (20 mg, 17.5 μmol, 0.04eq), the reaction was stirred vigorously at room temperature undernitrogen with monitoring by LC-MS. After three hours the reaction hadproceeded to approximately 50% conversion to desired product. Thereaction was concentrated and then partitioned between ethyl acetate(100 mL) and water (100 mL). The organic layer was then washed withwater (100 mL), brine (100 mL), dried over sodium sulfate, andconcentrated to dryness to provide crude aniline triflate 15. The crudeproduct was purified by flash chromatography, eluting with mixtures ofhexanes:ethyl acetate (60:40 to 30:70), to provide pure aniline triflate15 (118 mg, 25%). TLC: R_(f)=0.43, 25% hexanes in ethyl acetate. LC-MS:t_(R) 8.30 min, m/z (ES⁺) found 1088.2 (M+H)⁺.

(b)(S)-2-(4-aminophenyl)-8-((5-(((S)-2-(4-(3-(dimethylamino)propoxy)phenyl)-7-methoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H,11aH)-dione (16)

A flask was charged with aniline triflate 15 (118 mg, 109 μmol, 1 eq)dissolved in toluene (0.7 mL), ethanol (2.3 mL), and water (0.3 mL). Tothe stirred solution was added4-[3-(dimethylamino)propyloxy]phenylboronic acid pinacol ester (43 mg,142 μmol, 1.3 eq), sodium carbonate (53 mg, 0.5 mmol, 4.6 eq), andtetrakis(triphenylphosphine)palladium(0) (5 mg, 4.4 μmol, 0.04 eq), thereaction was stirred vigorously at room temperature under nitrogen withmonitoring by LC-MS. After four hours the reaction had reachedcompletion. The reaction was concentrated and then partitioned betweenethyl acetate (25 mL) and water (25 mL). The aqueous layer was extractedtwo times with ethyl acetate (25 mL). The organic layer was then washedwith water (50 mL), brine (50 mL), dried over sodium sulfate, andconcentrated to dryness to provide crude SEM dilactam 16. The crudeproduct was purified by flash chromatography, eluting with mixtures ofhexanes:ethyl acetate (50:50 to 0:100), to provide pure product 16 (78mg, 64%). TLC: R_(f)=0.38, 20% methanol in CH₂Cl₂. LC-MS: m/z (ES⁺)found 1117.8 (M+H)⁺. ¹H NMR (d7-DMF) δ (ppm) 0.00 (s, 18H), 0.90 (m,4H), 1.74 (m, 3H), 1.96 (m, 6H), 2.21 (s, 6H), 2.44 (t, J=7.2 Hz, 2H),3.25 (m, 2H), 3.62 (m, 4H), 3.80 (m, 2H), 3.96 (s, 6H), 4.11 (t, J=6.4Hz, 2H), 4.20 (m, 3H), 4.92 (m, 2H), 5.31 (dd, J=6, 10 Hz, 2H), 5.48 (m,4H), 6.73 (t, J=8.4 Hz, 2H), 7.01 (t, J=8.8 Hz, 2H), 7.29 (m, 3H), 7.40(m, 4H), 7.47 (m, 1H), 7.56 (t, J=8.4 Hz, 2H).

(c)(S)-2-(4-aminophenyl)-8-((5-(((S)-2-(4-(3-(dimethylamino)propoxy)phenyl)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11 aH)-one (17)

A flame-dried flask was charged with SEM dilactam 16 (70 mg, 63 μmol, 1eq) dissolved in anhydrous tetrahydrofuran (1.3 mL), and cooled to −78°C. Lithium triethylborohydride (0.13 mL of a 1 M solution in THF, 126μmol, 2 eq) was added dropwise and the reaction was stirred undernitrogen for 1.5 h, at which time LC revealed incomplete conversion toproduct. An additional 65 μL of reductant was added and the reaction wasstirred for one more hour. The reaction was quenched through theaddition of water (1 mL) and allowed to warm to room temperature, thendiluted brine (25 mL) and extracted three times with dichloromethane (25mL). The combined organics were washed with brine (25 mL), dried oversodium sulfate, and evaporated to dryness. The residue was dissolved ina mixture of dichloromethane (1.2 mL), ethanol (3.2 mL), and water (0.5mL), and silica gel (1.6 g) was added. The resulting slurry was stirredat room temperature for 4 days. TLC analysis revealed conversion toimine dimer 17, at which time the slurry was filtered over a sinteredglass funnel and the silica gel cake was washed with 10% methanol inchloroform until no further PBD absorbance was observed in the filtrate.Concentration of the filtrate provided crude imine dimer 17. Thematerial was dissolved in minimal dichloromethane and purified by radialchromatography on a 1 mm chromatotron plate eluted with CH₂Cl₂/MeOHmixtures (100:0 to 60:40) to provide 17 (31 mg, 60%). LC-MS: t_(R) 11.14min, m/z (ES⁺) found 825.4 (M+H)⁺.

Example 6

N-(4-((S)-8-((5-(((S)-2-(4-(3-(dimethylamino)propoxy)phenyl-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)phenyl-6-(2,5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl)hexanamide (18)

A flame-dried flask was charged with maleimidocaproic acid (5.2 mg, 25μmol, 1.5 eq) dissolved in 0.33 mL of 5% methanol in anhydrousdichloromethane. The acid was pre-activated by addition ofN-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (7.3 mg, 30 μmol, 1.8eq), followed by stirring at room temperature under nitrogen for 15minutes. The activated acid was then added to a flame-dried flaskcontaining PBD dimer 17 (13.5 mg, 16 μmol, 1 eq). The reaction wasstirred for 4 h at room temperature under nitrogen, at which time LC-MSrevealed conversion to product. The material was diluted indichloromethane and purified by radial chromatography on a 1 mmchromatotron plate eluted with CH₂Cl₂/MeOH mixtures (100:0 to 80:20) toprovide 18 (7.3 mg, 44%). LC-MS: t_(R) 9.09 min, m/z (ES⁺) found 1018.3(M+H)⁺.

Example 7

N—((S)-1-(((S)-1-((4-((S)-8-((5-(((S)-2-(4-(3-(dimethylamino)propoxy)phenyl-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)phenylamino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide(19)

A flame-dried flask was charged with maleimidocaproyl-valine-alaninelinker (Compound 36 in Example 13 of WO 2011/130613 A1) (9 mg, 24 μmol,1.5 eq) dissolved in 0.33 mL of 5% methanol in anhydrousdichloromethane. The acid was pre-activated by addition ofN-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (7.1 mg, 29 μmol, 1.8eq), followed by stirring at room temperature under nitrogen for 15minutes. The activated acid was then added to a flame-dried flaskcontaining PBD dimer 17 (13 mg, 16 μmol, 1 eq). The reaction was stirredfor 7 h at room temperature under nitrogen, at which time LC-MS revealedconversion to product. The material was diluted in dichloromethane andpurified by radial chromatography on a 1 mm chromatotron plate elutedwith CH₂Cl₂/MeOH mixtures (100:0 to 80:20) to provide 19 (5.1 mg, 27%).LC-MS: t_(R) 9.09 min, m/z (ES⁺) found 1188.4 (M+H)⁺.

Example 8

(a)2-(4-(2,5,8,11-tetraoxatridecan-13-yloxy)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(21)

To a mixture of 4-hydroxyphenylboronic acid pinacol ester (880 mg, 4mmol), bromomethyltetraethylene glycol (1.6 g, 6 mmol) and DMF (10 ml)was added Cs₂CO₃ (1.5 g; 8 mmol). The reaction mixture was stirred for˜65 h, and was poured into ethyl acetate (100 mL). The mixture waswashed with 0.1 N HCl (200 mL), water (3×100 mL) and brine (50 mL) andthe organic phase was dried over Na₂SO₄. Decanting and concentrationgave a brown oil which was purified on a 2 mm radial chromatotron plateeluting with 50% ethyl acetate in hexanes followed by 100% ethyl acetateto give 1.21 g (74%): NMR (d6-DMSO, 400 MHz) □ 7.59 (d, J=8.6 Hz, 2H),6.93 (d, J=8.6 Hz, 2H), 4.11 (t, J=4.7 Hz, 2H), 3.73 (m, 2H), 3.60-3.45(m, 14H), 3.41 (m, 2H), 3.23 (s, 3H), 1.27 (s, 12H); LC-MS: m/z (ES+)found 433.64 (M+Na)⁺.

(b)(R)-2-(4-(2,5,8,11-tetraoxatridecan-13-yloxy)phenyl)-8-((5-(((R)-2-(4-aminophenyl)-7-methoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H,11aH)-dione(22)

To a mixture of the monotriflate 15 (200 mg, 0.18 mmol) and TEG boronateester (111 mg, 0.27 mmol) in a mixture of toluene (2 mL) and ethanol (1mL) was added 2M Na₂CO₃ (0.5 mL) and tetrakis Pd (6 mg, 0.054 mmol).After stirring at room temperature for 3 h, the reaction mixture waspoured into ethyl acetate, washed with water (3×50 mL) and brine (50 mL)and was dried over Na₂SO₄. The solution was decanted, concentrated andthen purified on a 2 mm radial chromatotron plate eluting with 5%methanol in dichloromethane. This gave 207 mg (94%) as a yellow solid:¹H NMR (CDCl₃, 400 MHz) □ 7.39-7.34 (m, 7H), 7.26 (m, 2H), 6.89 (d,J=9.0 Hz, 2H), 6.66 (d, J=8.6 Hz, 2H), 4.70 (dd, J=10.2, 2.0 Hz, 2H),4.18-4.03 (m, 8H), 3.93 (s, 3H), 3.90-3.62 (m, 21H), 3.55 (dd, J=5.1,3.2 Hz, 2H) 3.38 (s, 3H), 3.12 (pent, J=5.0H, 2H), 2.05 (m, 6H), 1.72(m, 2H), 1.0 (m, 4H), 0.3 (s, 18H); LC-MS: m/z (ES+) found 1222.98(M+H)⁺.

(c)(R)-2-(4-(2,5,8,11-tetraoxatridecan-13-yloxy)phenyl)-8-((5-(((R)-2-(4-aminophenyl)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one(23)

To a mixture of the SEM dilactam (207 mg, 0.17 mmol) in THF (10 mL) at−78° C. was added Superhydride (LiHBEt₄ as a 1N solution in THF, 0.34mL, 0.34 mmol). The reaction mixture was stirred for approximately 2 h,at which time LC-MS analysis of the reaction mixture revealedapproximately 50% conversion to fully reduced SEM-carbinolamineintermediate, with mono-reduced material still present. An additionalaliquot of Superhydride (0.34 mL, 0.34 mmol) was added and the reactionwas stirred at −78° C. for an additional hour. LC-MS inspection revealedthat the reaction still had not progressed to completion, therefore athird aliquot of Superhydride (0.34 mL, 0.34 mmol) was added and thereaction mixture was placed in a −80° C. freezer for 16 hours. Thereaction mixture was then quenched with water (5 mL), allowed to warm toan ambient temperature and was poured into ethyl acetate (100 mL). Afterwashing with brine, the mixture was dried over Na₂SO₄. The organic phasewas decanted, concentrated under reduced pressure, and then treated witha mixture of ethanol (14 mL) dichloromethane (5 mL), waster (7 mL) andsilica gel (5 g) for 72 hours. The mixture was filtered through afritted glass funnel and washed several times with 10% methanol indichloromethane, before the solution was concentrated under reducedpressure. The mixture was purified on a 2 mm radial chromatotron plateeluting with 5% methanol in dichloromethane to give 46 mg (29%): LC-MS:m/z (ES+) found 930.85 (M+H)+.

(d)N—((S)-1-(((S)-1-((4-((R)-8-((5-(((R)-2-(4-(2,5,8,11-tetraoxatridecan-13-yloxy)phenyl-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-6-(2,5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl)hexanamide (24)

To a mixture of the mc-val-ala-OH (50 mg, 0.129 mmol) in 5% methanol indichloromethane (1 mL) at 0° C. was added EEDQ (32 mg, 0.129 mmol). Themixture was stirred for 15 min, and then a solution of the aniline (44mg, 0.044 mmol) in 5% methanol in dichloromethane (1 mL) was added. Thereaction mixture was allowed to stir for 3 hours, was diluted withdichloromethane (2 mL) and was aspirated directly onto a 2 mm radialchromatotron plate. The product was eluted with a gradient of 2.5% to 5%methanol in dichloromethane to give 22.5 mg (40%): LC-MS: m/z (ES+)found 1294 (M+H)⁺.

Example 9 Preparation of PBD Dimer Conjugates

Antibodies with introduced cysteines: Antibodies to CD70 containing acysteine residue at position 239 of the heavy chain were fully reducedby adding 10 equivalents of TCEP and 1 mM EDTA and adjusting the pH to7.4 with 1M Tris buffer (pH 9.0). Following a 1 hour incubation at 37°C., the reaction was cooled to 22° C. and 30 equivalents ofdehydroascorbic acid were added to selectively reoxidize the nativedisulfides, while leaving cysteine 239 in the reduced state. The pH wasadjusted to 6.5 with 1M Tris buffer (pH 3.7) and the reaction wasallowed to proceed for 1 hour at 22° C. The pH of the solution was thenraised again to 7.4 by addition of 1 M Tris buffer (pH 9.0). 3.5equivalents of the PBD drug linker in DMSO were placed in a suitablecontainer for dilution with propylene glycol prior to addition to thereaction. To maintain solubility of the PBD drug linker, the antibodyitself was first diluted with propylene glycol to a final concentrationof 33% (e.g., if the antibody solution was in a 60 mL reaction volume,30 mL of propylene glycol was added). This same volume of propyleneglycol (30 mL in this example) was then added to the PBD drug linker asa diluent. After mixing, the solution of PBD drug linker in propyleneglycol was added to the antibody solution to effect the conjugation; thefinal concentration of propylene glycol is 50%. The reaction was allowedto proceed for 30 minutes and then quenched by addition of 5 equivalentsof N-acetyl cysteine. The ADC was then purified by ultrafiltrationthrough a 30 kD membrane. (Note that the concentration of propyleneglycol used in the reaction can be reduced for any particular PBD, asits sole purpose is to maintain solubility of the drug linker in theaqueous media.)

Example 10 Determination of In Vitro Activity of Selected Conjugates

The in vitro cytotoxic activity of the selected antibody drug conjugatewas assessed using a resazurin (Sigma, St. Louis, Mo., USA) reductionassay (reference: Doronina et al., Nature Biotechnology, 2003, 21,778-784). The antibody drug conjugate was prepared as described above.

For the 96-hour assay, cells cultured in log-phase growth were seededfor 24 h in 96-well plates containing 150 μL RPMI 1640 supplemented with20% FBS. Serial dilutions of ADC in cell culture media were prepared at4× working concentration; 50 μL of each dilution was added to the96-well plates. Following addition of ADC, the cells were incubated withtest articles for 4 days at 37° C. Resazurin was then added to each wellto achieve a 50 μM final concentration, and the plates were incubatedfor an additional 4 hours at 37° C. The plates were then read for theextent of dye reduction on a Fusion HT plate reader (PackardInstruments, Meridien, Conn., USA) with excitation and emissionwavelengths of 530 and 590 nm, respectively. The IC₅₀ value, determinedin triplicate, is defined here as the concentration that results in a50% reduction in cell growth relative to untreated controls.

Referring to the table below, the in vitro cytotoxicity of the ADC usingthe 96 hour assay is shown. The ADC was tested against antigen positiveand antigen negative cell lines.

h1 F6 is the humanized anti-CD70 antibody described below.

In Vitro Activity

TABLE 1 IC₅₀ in pM following 48 hours treatment compound 786-O Caki-1HL60 HEL9217 17 7 2 3 2 23 100 100 40 100

TABLE 2 IC₅₀ in pM following 96 hours treatment antigen-negative ADCdrugs/Ab 786-O Caki-1 cell line h1F6ec-6 1.8 260 12 28,000 h1F6ec-9 1.5120 4.9 60,000 h1F6ec-14 1.8 380 24 20,000

TABLE 3 IC₅₀ in pM following 96 hours treatment antigen-negative ADCdrugs/Ab 786-O Caki-1 UMRC3 cell line h1F6ec-18 1.9 540 63 1000 4000h1F6ec-19 1.9 13 3.8 25 2000 h1F6ec-24 2.0 30 8 — 3000

Example 11 Determination of In Vivo Cytotoxicity of Selected Conjugates

All studies were conducted in accordance with the Animal Care and UseCommittee in a facility that is fully accredited by the Association forAssessment and Accreditation of Laboratory Animal Care. ADC tolerabilitywas first assessed to ensure that the conjugates were tolerated at thedoses selected for the xenograft experiments. BALB/c mice were treatedwith escalating doses of ADC formulated in PBS with 0.5 M arginine and0.01% Tween 20. Mice were monitored for weight loss and outward signs ofmorbidity following treatment; those that experienced greater than 20%weight loss or displayed signs of morbidity were euthanized. Theantibody used was a CD70 antibody, humanized h1F6 IgG1 (WO2006/113909incorporated by reference in its entirety and for all purposes), with apoint mutation substituting cysteine for serine at position 239.Conjugation to the Drug Unit is through the introduced cysteine atposition 239. An average of 2 drugs is loaded per antibody.

In vivo therapy experiments were conducted in xenograft models in micebearing CD70+renal cell carcinoma. Tumor (Caki-1) fragments wereimplanted into the right flank of Nude mice. Mice were randomized tostudy groups (n=5 (786-0) with each group averaging around 100 mm³. TheADC or controls were dosed ip according to the schedule indicated. Tumorvolume as a function of time was determined using the formula (L×W²)/2.Animals were euthanized when tumor volumes reached 1000 mm³. Miceshowing durable regressions were terminated around day 100 post implant.

Referring to FIG. 1, the results of a treatment study using anh1F6-compound 18 and h1F6-compound 19 conjugate in a CD70+renal cellcarcinoma model are shown. Dosing was carried out at q7d×2. In theFIGURE,

is untreated, ▪ is treatment with h1F63c-18 at 0.1 mg/kg, □ is treatmentwith h1F63c-18 at 0.3 mg/kg, ▴ is treatment with h1F63c-19 at 1 mg/kg, Δis treatment with h1F63c-19 at 3 mg/kg,

Results of a mouse tolerability experiment an h1F6-compound 6 conjugatedemonstrated that a single dose of ADC at 1 mg/kg was well toleratedwith no signs of weight loss or morbidity out to 30 days. Administrationof a higher dose (2.5 mg/kg) resulted in weight loss.

1. A compound, or a pharmaceutically acceptable salt thereof, selectedfrom the group consisting of:

wherein: (a) R¹⁰ is H, and R¹¹ is OH, OR^(A), where R^(A) is saturatedC₁₋₄ alkyl; (b) R¹⁰ and R¹¹ form a nitrogen-carbon double bond betweenthe nitrogen and carbon atoms to which they are bound; or (c) R¹⁰ is Hand R¹¹ is SO_(z)M, where z is 2 or 3 and M is a monovalentpharmaceutically acceptable cation, or both M together are a divalentpharmaceutically acceptable cation.
 2. A compound according to claim 1,wherein R¹⁰ and R¹¹ and form a nitrogen-carbon double bond.
 3. Acompound selected from the group consisting of:

wherein either (a) R¹⁰ is carbamate nitrogen protecting group, and R¹¹is O-Prot^(◯), wherein Prot^(◯) is an oxygen protecting group; or (b)R¹⁰ is a hemi-aminal nitrogen protecting group and R¹¹ is an oxo group.4. A compound according to claim 3, wherein R¹⁰ is Troc and/or R¹¹ isOTBS.
 5. A compound according to claim 3, wherein R¹¹ is oxo and R¹⁰ isSEM.
 6. A compound according to claim 2, which is of formula C1:

wherein R¹⁰ and R¹¹ and form a nitrogen-carbon double bond.
 7. Acompound according to claim 2, which is of formula C2:

wherein R¹⁰ and R¹¹ and form a nitrogen-carbon double bond.
 8. Acompound according to claim 2, which is of formula C3:

wherein R¹⁰ and R¹¹ and form a nitrogen-carbon double bond.
 9. Acompound according to claim 2, which is of formula C4:

wherein R¹⁰ and R¹¹ and form a nitrogen-carbon double bond.
 10. Acompound according to claim 2, which is of formula C5:

wherein R¹⁰ and R¹¹ and form a nitrogen-carbon double bond.
 11. Acompound according to claim 2, which is of formula C6:

wherein R¹⁰ and R¹¹ and form a nitrogen-carbon double bond.